Ca

VERSITYO?

AND SJV"

* .uRAL

IMENT P

BURLINGTON.

BULLETIN NO. 167

JUNE, 1912

I ' 506479

— — I* . A- . So

Qnp

Burlington:

Free Press Pki.vhno Co.,

L912.

ROL

' i ATH

. HILL-.
P. A. RICH, Veterinarian.
CASSIUS PECK, Farm Si
C. H. JONES, Chemist.
» F HAWES, (State Forester), Foresiei
S, Horticulturist.
Plant Pathologist.
)tanist.

i, Animal and Dairy Husbandman.
P. A. ii£,i>^~--r, Assistant Chemist.
JENNIE L. ROWELL, Assistant Chemist.
W. B. DERBY, Assistant Chemist.
G. C. CUNNINGHAM, Assistant Plant Pathologist.
**■ *c LOMBARD, Assistant Horticulturist.
C. W. U rpeNTER, Assistant Bacteriologist.
W. J. LAMt^nuGH, Computer.
W. F. HAMMOND Expert in Horse Breeding.*
STANLEY HARGK^ ■• VES, Gardener.
ADELLE ORTON, stenogu ^her.
C. P. SMITH. Treasurer.

fts*Copies of the reports an«*4 bulletins of
free of charge to any address upon application.

*3"Address xnmnnicarions nc^

the Ex^

Leal, horticultural
at the head of Main str
Glories are at Williams Science 1
aiy laboratories at 499 Main Street.
^i-m and buildings are on the Williston road, ad
diversity grounds on the east.

"la cooperation with Hu. An. Ind., U. B. Dept, Agr.

BULLETIN 167: MICRO-ORGANISMS 01 MAPLE SAP.

Part I. Micro-organisms Occurring in Maple Sap and their
Influence on the Color, Flavor and Chemical ComposU
tion of Sirup.

By II. A. Edson

Fart II. Discussion of Physical and Chemical Data Secured
on Maple Sirups Obtained from Saps Inoculated with
— Micro-organisms.

By C. I J. Jones

Part III. Technical Description of Certain Bacteria Occur=
ring in Maple Sap.

A. Description of Bacillus aceris (n. sp.).

B. Brief Description of the Pink Cocci of Maple Sap.

C. The Green Fluorescent Bacteria Occurring in Maple

Sap.

By H. A. Edson and C. W. Carpenter

The completion of the studies reported in Tart I and in
Part HI (A) of this bulletin was made possible through the
courtesy of the Chief of the Bureau of Plant Industry of the
United States Department of Agriculture. The author of those
parts, the former station bacteriologist, when he withdrew from
station employ to enter that of the Department, had not pursued
the investigation far enough to warrant final publication. The
Chief of the Bureau generously allowed him to continue his
work after he had become connected with the Department dur-
ing the succeeding sugar season, at intervals during the year,
and for several months early in [912 while preparing the mat-
ter for the press. Had it not been for this cordial cooperation,
the matter could hardly have been brought to a successful issue ;
and the Station gratefully acknowledges its obligation for the
courtesy.

J. L. Hills, Director.

324 Bulletin 167

TABLE OF CONTENTS

Part I. Micro-organisms Occurring in Maple Sap and their In-
fluence on the Color, Flavor and Chemical Composi-
tion of Sirup 333-418

Introduction 333

The relation of micro-organisms to spoiled maple sap 336

The number of micro-organisms in maple sap 338

Number of organisms in sour maple sap 339

Table 1. Organisms per cc. in sour maple sap 339

Number of organisms in sweet maple sap 340

Table 2. Organisms per cc. in sweet maple sap 340

Table 3. Organisms per cc. in sap obtained in sterilized

containers 341

Bacteria] content of sap obtained under septic conditions as

compared with aseptic conditions 341

Table 4. Organisms in sap from fresh and from sour tap-
holes 341

Influence of tap-hole infection upon the flora of sap 342

• Table 5. Organisms from tap-hole and spout per cc. of

maple sap 342

Influence of container on bacterial flora 343

General plan of field experiments 343

Method of sirup scoring :'>4~>

Table 6. Amounts of ingredients to be used in preparing

solutions for the color scale 348

Table 7. Values assigned to color grades 349

Table 8. Values assigned to flavor grades 349

Inoculation experiments in 1909 349

Detailed discussion of samples :;:>1

Series 1: Sirups 1 to G 35]

Series 2: Sirups 7 to 12 353

Series 3 : Sirups 13 to 20 355

Sirups from last run sap in 1909 357

Inoculation experiments in 1910 359

Series 4: Sirups 27 to 40 361

Series 5: Sirups 41 to 48 3G5

Series G: Sirups 49 to 37

Series 7 : Sirups 58 to 65 370

Influence of container upon quality of sirup 372

Series 8 : Sirups G8 to 73 373

Series 9: Sirups 74 to 81 75

Micro-organisms of" Maple Sap 325

Sugar and sirup from sour sap :!77

Sirup from last run sap in 1910 :'.7'.t

Inoculation experiments in 1911 3N0

Series 10: Sirups 89 to 9G 381

Series 11: Sirups 97 to 104 383

Series 12 : Sirups 1 05 to 11 2 385

Series 13: Sirups 113 to 120 386

Sirups from last run sap in 1911 :'.s<i

Statistical summary of field experiments 391

Table 9. Tabular summary of statistical data 392-397

Discussion of related sirups in groups 398

Table 10. Statistical tables showing related samples in

juxtaposition 102-409

Table 11. Summary of averages of related groups in

order of score 410- 1 1 I

Comparison of sirups made from inoculated sap and from

natural sour sap 412

Discussion of depreciation values 413

Table 12. Order of average depreciation in color 413

Table 13. Order of average depreciation in flavor 414

Table 14. Order of average depreciation in score 415

Discussion of last run sirups 415

Influence of inert extraneous material 415

Conclusions 416

Remedial measures 417

Part II. Discussion of Physical and Chemical Data Secured on
Maple Sirups Obtained from Saps Inoculated with

Micro-organisms 419-474

Introduction 419

Table 15. Moisture contents of sirup samples 420

Explanation of physical and chemical data shown in tables

16 to 34 421

Group 1. Controls 1l>1

Table 16. Maple sirup. Controls 422

Table 17. Minimum ash and malic acid values for pure

maple sirup 423

Group 2. Incubator controls 425

Table IS. Maple sirup. Incubator controls 426

Group 3. Inoculated with non-fluorescent bacteria 427

Table 19. Maple sirup. Inoculated with non-fluorescent

bacteria Us

326

Bulletin [()?

Group 4. Inoculated with pink cocci 4 l!rt

Table 20. Maple sirup. Inoculated with pink cocci 430

Group 5. Failures 431

Table 21. Maple sirup. Failures 432

Group 6. Inoculated with red yeasts 4:!:;

Table 22. Maple sirup. Inoculated with red yeasts 434

Group 7. Inoculated with gray yeasts 435

Table 23. Maple sirup. Inoculated with gray yeasts.... 43G

Group 8. Inoculated with fluorescent bacteria 437

Table 24. Maple sirup. Inoculated with fluorescent bac-
teria 438

Group 9. Composites 440

Table 25. Maple sirup. Composites 441

Group 10. Inoculated with Bacillus aceris 442

Table 26. Maple sirup. Inoculated with Bacillus aceris. . 443

Group 11. Last run, sweet 444

Table 27. Maple sirup. Last run, sweet 445

Group 12. Inoculated with fluorescent and other organ-
isms and with spore-bearers 446

Table 28. Maple sirup. Inoculated with fluorescent and

other organisms and with spore-bearers 447

Group 13. Inoculated with green molds 448

Table 29. Maple sirup. Inoculated with green molds.... 449

Group 14. Tin vs. wooden buckets 150

Table 30. Maple sirup. Tin vs. wooden buckets 451

Group 15. Inoculated with pink yeast; burned control.. 452
Table 31. Maple sirup. Inoculated with pink yeast;

burned control 453

Group 16. Last run, sour 454

Table 32. Maple sirup. Last run, sour 455

Group 17. Sour sap, kept 456

Table 33. Maple sirup. Sour sap, kept 457

Summary of averages secured on the sundry groups dis-
cussed 458

Table 34. Average analyses of the maple sirup groups... 459

Invert sugar content of maple sirup 461

Table 35. Average invert sugar content of the different
groups and maximum and minimum in indi-
vidual samples 462

Discussion of the total and insoluble ash and malic acid

values 463

Micro-organisms of Maple Sap 321

Table 36. Sirups deficient in total ash, insoluble ash, or

malic acid value 464

Table 37. Number of samples and percent below standard
in total ash, insoluble ash and malic acid

value 467

Table 38. Number and nature of deficiencies 4f,s

Table 39. Deficiencies grouped according to sample num-

oers 169

Table 40. Effect of diluting, boiling and filtering on con-
centrated maple sirup compared with the

original analyses of the same samples 471

Summary 473

Part III. Technical Description of Certain Bacteria Occurring

in Maple Sap 475-602

A. Description of Bacillus aceris (n. sp.) 475-516

Summary of characters 475

Occurrence and character 475

Morphology 475

Cultural features 476

Physical and biochemical features 477

Detailed description 478

Occurrence 478

Form 479

Morphological characters 480

Dimensions 480

Cultures on agar hanging blocks 480

Fission 481

Grouping 4.81

Motility and flagella 481

Spores 482

Capsule 482

Involution forms 483

Staining reactions . . 483

Cultural characters 4N4

Methods 484

Agar stroke 484

Agar stab 484

Agar plates 485

Carbohydrate agars 4s.",

Gelatin 486

Gelatin stroke 486

Bulletin 167

Gelatin stab 486

Gelatin colonies 4^7

Broth 4S7

Potato blocks 488

Milk 488

Acid production in milk 488

Table 41. Acid production in milk * . . . 489

Table 42. Average acid production in milk in cc. N/1

per liter 490

Litmus milk 491

Starch jelly 491

Cohn's solution 491

Uschinsky solution 491

Fermi's solution 492

Silicate jelly 492

Sodium chlorid in bouillon 492

Growth in bouillon over chloroform 492

Maple sap sterilized 492

Insterilized sap 493

Artificial maple sap 493

Physical and biochemical features 494

Gas production in milk 494

Carbohydrate broth 4^4

Table 43. Acid production in carbohydrate broths.... 49G

Table 44. Gas production in carbohydrate broths 498

Table 45. Proportion of carbon dioxid to total gas.... 4'.'^

Fermentation of potato starch 499

Gas production in modified Uschinsky solution contain-
ing carbohydrates 499

Ammonia production 500

Table 46. Ammonia produced in nutrient, broth 500

Nitrate reduction 500

Indol production 501

Production of phenol 501

Hydrogen sulphid production 502

Toleration of acids 502

Toleration of sodium hydroxid

Optimum reaction 504

Temperature relations 505

Growth in carbon dioxid atmosphere 505

Desiccation

Micro-organisms of Maple Sap :'-•-".,

Insolation 507

Production of alcohol 507

Production of proteolytic enzyms 509

Production of diastatic ferments 510

Production of invertase 510

Effect of germicides 512

Formaldehyde 512

Phenol 513

Nitrogen requirements 514

Series A. Glycerin •". I :,

Series B. Mannit r, 1 r.

Series C. Dextrose 515

Table 47. Growth on nitrogen media 515

Name 516

B. Brief Description of the Pink Cocci of Maple Sap 516-521

Morphology 516

Cultural characters 517

Physical and biochemical features 519

Table 48. Acid production on carbohydrate broths.... 520

C. The Green Fluorescent Bacteria Occurring in Maple Sap. . . .521-602

Introduction 521

Preliminary studies upon 42 strains of green fluorescent

sap bacteria 523

Isolation r>2::

Methods of work 525

Nutrient broth 526

Agar medium 526

Gelatin medium 526

Rejuvenation and stock 526

Detailed features of the forty-two strains 527

Morphology 527

1. Form 527

2. Grouping 527

3. Motility 527

4. Endospores 527

5. Capsules 52s

6. Stains 528

Aqueous anilin stains 528

Cultural features 52s

1. Agar stroke 528

2. Potato 528

330

Bulletin 167

3. Agar stab 528

4. Gelatin 528

5. Nutrient broth 529

6. Milk 529

7. Litmus milk 529

8. Gelatin colonies 530

9. Agar colonies 530

10. Cohn's nutrient solution * . . 530

1 1. Uschinsky solution 531

12. Nitrogen requirements 531

Physical and biochemical features 532

1. The action of the organisms upon dextrose, lactose,

sucrose and glycerin 53J

Table 49. Acid and alkali produced in sugar free nutri-
ent bouillon 534

Table 50. Acid and alkali produced in 2% dextrose

bouillon 535

Table 51. Acid and alkali produced in 2</< dextrose

bouillon 53G

Table 52. Acid and alkali produced in 2% lactose

bouillon 537

Table 53. Acid and alkali produced in 2f/o lactose

bouillon 538

Table 54. Acid and alkali produced in 2% sucrose

bouillon 539

Table 55. Acid and alkali produced in 5% glycerin

bouillon 540

Table 50. Acid and alkali produced in 5% glycerin

bouillon 511

2. Ammonia production 542

Table 57. Ammonia production 54:'

Table 58. Ammonia production 51::

3. Action on nitrates ."it:;

4. Indol 544

5. Temperature relations 544

6. Acids and alkalies 544

7. Reduction processes 545

8. Odor 545

9. Gas production 545

10. Crystals 545

11. Oxygen requirements 545

Micro-organisms of Maple Sap 331

12. Fluorescence 546

13. Diastasic action on potato starch 548

14. Group numbers 548

15. Separation of the 42 strains into groups 548

Chart showing the separation of the 42 strains into

groups -. 550

Comparative studies of seven representative strains of green

fluorescent sap bacteria and six known species 552

Detailed features ."..">:;

Morphology 553

1 . Vegetative cells 553

Table 59. Limits of size on hanging block 553

Table GO. Limits of size on hanging drops 553

Table 61. Limits of size on stained preparations 554

2. Endospores 554

3. Flagella 554

4. Capsules 555

5. Zoogloea ;,:,i;

6. Involution forms 556

7. Staining reactions 556

Cultural features 557

1 . Agar stroke 557

2. Potato slants \ 558

3. Loeffler's blood serum 558

4. Agar stab 558

"». Gelatin stab at 20° C 559

6. Nutrient broth 563

7. Milk 564

Table 62. Acid production in milk 564

8. Litmus milk ;,i;i;

9. Gelatin colonies 567

10. Agar colonies 568

1 1. Cohn's nutrient solution r>70

1 2. Uschinsky solution ,",71

Table 63. Uschinsky solution 572

Table 64. Modified Uschinsky solution 7.73

13. Nitrogen -.74

Table 65. Nitrogen requirements :,74

Table 66. Nitrogen requirements 7,7;,

Physical and biochemical features 7,71;

1 . Sugar free medium 7,7c

332 Buixetin 167

Table 67. Percent normal acid or alkali produced in

sugar free medium 577

2. Sugar media 577

Table 68. Percent normal acid or alkali produced on

dextrose peptone; lactose peptone; sucrose pep-
tone; ; glycerin peptone "77

3. Action on nitrates in nitrate broth 579

4. Indol 580

Table 69. Indol production in nutrient broth 580

Table 70. Indol production in Dunham's peptone 5S1

f>. Toleration of acids and alkalies 581

Table 71. Growth on acid and alkali media after 24

hours 582

Table 72. Growth on acid and alkali media after 3

days 583

6. Optimum reaction for growth in bouillon 583

Table 73. Optimum reaction 584

7. Vitality on culture media 584

8. Temperature relations; thermal death point 584

Table 74. Thermal death point 585

9. Drying ~'s"

Table 75. Resistance to desiccation 586

10. Insolation 586

Table 76. Influence of insolation 587

Table 77. Influence of insolation 587

11. Acids produced 588

12. Alkalies produced 58S

13. Crystals 588

14. Diastasic action on potato starch 588

15. Anaerobiosis 588

Group number of the 13 strains 590

Species 590

New species, strain CXV 593

Brief description of Bacillus parallel us (n. sp.) 594

Morphological characters 594

Cultural features 594

Physical and biochemical features 596

Table 78. Brief characterization of the 12 strains of

Ps. fluoresceins 598

Conclusions 59S

Bibliography (Part III C) 600

Index (in:1,

PART T

MICRO-ORGANISMS OCCURRING IN MAPLE SAP AM) THEIR IN-
FLUENCE ON THE COLOR, FLAYOH AM) CHEMICAL
COMPOSITION OF SMUT

By H. A. Edson

I NTRODUCTION

A preliminary contribution upon the work presented in this
paper appeared in bulletin 151 (1910). Only the more important
facts reported at that time are restated in the present paper, but
quotations and repetitions stripped of details are introduced when
essential to a clear and complete presentation. The problem is
most satisfactorily introduced, however, by a somewhat lengthy
quotation from the former paper, in which, for the information
of any who may not be familiar with them, are included brief
descriptions of the main facts of sap flow and of sugar making" as
practiced in Vermont.

"Late in March, in this section, evidences of coming spring-
appear. The nights are still cold and frosty but the days are
genial and the temperature rises a few degrees above the freez-
ing point. If, at this time, the trunk or limbs of certain species
of the genus Acer are fresh wounded a sweet sap exudes. The In-
dians were familiar with this phenomenon before white men
came ; and had learned to collect, to concentrate and to make
sugar from this sap. The early settlers1 learned from them the
essential steps which, in modified form, constitute the procedure
followed in the maple sugar industry today."

"According to modern practice the tree is tapped by boring
a half-inch hole 2 inches deep about 4 feet from the ground. A
round, hollow spout or "spile" of wood or metal, upon which
is suspended a bucket to catch the dripping sap, is driven into
the hole. The sap flow is not continuous but is divided into
short intermittent periods, technically termed 'runs.' It oc-
curs only during the three or four weeks which immediately
precede the unfolding of the leaf buds. Both its periodicity and

1 Garden and Forest 4, p. 171.

334 Bulletin 167

its duration depend upon weather conditions. The sap is more
likely to flow in the daytime than at night; and the more im-
portant runs are confined to what are spoken of as 'good sap
days.' These occur only after the air temperature has remained
below freezing for some time. If, following such a cold spell,
the temperature rises materially above 320 F. a good run is like-
ly to ensue. Excessive warmth and high winds check the flow.
Freezing nights followed by moderately warm, cloudy days, and
the absence of excessive sunshine and heavy winds, are the
meteorological conditions which characterize the best sugar
weather. So long as the air temperature remains essentially
constant, whether warm or cold, little or no sap is obtained."

"The buckets in which the sap is caught are made of wood,
tin, or galvanized iron; and, in the better works, are covered to
keep out rain, snow and other foreign material. The sap is col-
lected after each day's run ai?d taken to the boiling house, tech-
nically known as the sugar house where it is concentrated into
sirup in large shallow pans over a n?.arui§' wood fire as rapidly
as the capacity of the equipment will permit."

"Maple sap is a sweet liquid containing a varying amount,
averaging from 2 to 3%, of sucrose, and, usu^11^ traces of in-
vert sugar. In addition to these carbohydrates it-- contains small
amounts of proteids, of mineral matter, mainly lime\and Potasll>
and of acids, mainly malic acid. The sap of the earliei" flows is
water clear and transparent, and possesses a clean, sweet /avor-
With the advance of the season, however, it undergoes a ma\Orked
change. As the days grow warmer and night freezes are k°ss
severe and less frequent, the sap gradually becomes cloudy am 1
discolored and unpleasant flavors develop. Such sap 'while
usually containing only the normal amount of acid, is popularlv
termed -sour.' It rapidly deteriorates when stored even for
a few hours. Several types of sour sap are recognized by suw
makers, to which the descriptive terms 'milky,' 'stringy ' 'red '
and. particularly, 'green" are commonly applied. Green' sap is
almost always secured just before the close of the season, when

M [< R0 ORGANISMS "1 M \n.i-. SAP

the leaf buds are ready to open. It is popularly believed thai

the swelling of the buds, associated with the renewal of vegeta-
tive activity in the tissues of the tree, is accompanied by a
change in the composition of the sap within the trunk ; and that
the alteration in color and flavor are manifestations of this
change. The term 'buddy' is universally used to descrihe this
sort of sap." (See Plate V).

"The sirup made from late runs is much inferior to that
derived from the earlier flows. 'Last run' goods are often
very dark in color and usually lack the delicate flavor possessed
by the hest sirups. Moreover, the quality of sirup varies
markedly from year to year and these variations are seldom local
in distribution. Such widespread fluctuations in quality are not
accidental but of necessity must be associated with some funda-
mental cause or causes. It is conceivable that they may be re-
lated to weather conditions, either during the preceding summer
or during the progress of the sugar season. It is known, more-
over, that inferior products result from carelessness and lack
of cleanliness in collecting and handling maple sap. Such pro-
cedures must occasion a great increase in the bacterial content
of the sap, just as in dairying they entail serious bacterial con-
tamination in milk. The proteid, carbohydrate and mineral con-
tents of maple sap are sufficient to make it a fairly good medium
for the development of bacterial life, provided suitable tempera-
ture relations are maintained ; and the vital activities of large
numbers of micro-organisms would presumably affect the flavor
and quality of the sirup produced under such conditions."

"Reflection upon these facts strongly suggests the pos-
sibility that micro-organisms may be associated with the inferi-
ority of the maple output in all the cases cited, or that, indeed,
they may be the direct cause of the troubles. Inevitably they
must be present in the sap and it is to be expected that they
would be more abundant toward the close of the sugar season
than earlier, because the warmer weather would favor their in-
creasingly rapid development and multiplication. It is conceiv-

336 Bulletin 167

able that the 'off' seasons may prove to he those wherein con-
ditions foster this microscopic life to an unusual degree. More
favorahle temperature relations, or longer periods of incubation,
or both, in 'off' years and in the latter part of the season, might
reasonably be expected to promote the multiplication of organ-
isms in the tap-hole, spout and bucket, thus producing heavy in-
itial inoculation of the sap. Uncleanly methods would certainly
result in this condition, and in any ease, storing, even for a
short length of time, would serve to increase the troubles due
to the vital activities of microscopic organisms, particularly if the
temperature of the storage house was considerably above the
freezing point, as is apt to be the case."

The experimental work upon which a report is submitted
in these pages was undertaken in an effort to determine as far
as might be possible the answers to the following questions:
What is the cause of the deterioration of maple sap? Is it
due to changes in composition occurring within the tissues of
the tree as a result of the resumption of protoplasmic activities
and vegetative vigor? Is it due to the action of micro-organ-
isms in the sap after it leaves the vascular bundles of the trunk?
Or is it to be attributed to a combination of these causes?

THE RELATION OF MICRO-ORGANISMS TO SPOILED MAPLE SAP
The early studies, previously reported, have shown that
maple sap while in the vascular bundles of the tree is sterile, and
that when drawn without contamination and stored under aseptic
conditions it keeps perfectly for years, even with free access of
air. Hence the changes in sap known as souring are not t<>
be attributed to the action of enzyms present as natural in-
gredients or to other forms of auto-decomposition. Careful
studies of the microscopic flora of spoiled sap have shown that
micro-organisms are constantly associated with the process of
souring.

In certain types of spoiled sap specific groups of organisms
are likely to predominate, while in other types several of the

Micro-organisms oi? Maple Sap :i:'>7

common forms may be found growing together in association. In
addition to various common saprophytic bacteria, wild yeasts of
several types, and green molds of the genera Penicillium and
Knrotium arc frequently met with, particularly towards the cl<
of the season when the temperature becomes more favorable
for their development. Green sap. for example, is associated
with the presence of a group of bacteria characterized by green
fluorescence and does not appear to be caused by the swelling
of the buds to which it is so frequently related in time, hi one
type of stringy sap occasionally found, the only significant or-
ganism was one described in this paper as Bacillus a ecu's { new
Species), while in another type of stringy sap common at the
close of the season, several species of bacteria, various wild
yeasts, and molds of different genera occurred in association.
Gray and red yeasts are commonly associated with bacteria in
so-called red sap. Spore-bearing bacteria of the hay bacillus
type were frequent in certain samples of milky sap examined.
while the condition of other samples seemed attributable to the
collective action of many different organisms.

The conclusion stated in the preliminary publication, that
there is a causal relation between micro-organisms and sour
sap and the resulting poor sirup, has been confirmed by all the
examinations made during the progress of the work, and by the
inoculation experiments to be reported later. A sequence of
species was noted during the season of 1909, and discussed on
page 494 of bulletin 151 where the following sentences occur:
"The predominating organisms found during the early days of
the sugar season belong to the yeast-like group, while bacteria
were relatively few in numbers. As the season advanced these
conditions were reversed." Subsequent studies have not con-
firmed this observation. For the past two years relatively few
organisms of any kind have appeared on the plates early in
the season, but as a rule bacteria have predominated, anjd in
general yeast-like organisms have appeared in important num-
bers only after the season was well advanced.

338 Bulletin 167

tup; number oe micro-organisms in maple SAr
During the first two seasons covered by these studies (1907-
and 1908) no attempt was made to determine the bacterial count
in maple sap of different types. Attention was rather directed
to determining the predominating forms of micro-organisms
present, and to securing cultures to be employed as a basis for
the morphological and biochemical studies and for inoculation
work, which have constituted the major portion of the problem.
In connection with the field studies of the past three years, how-
over, some attention has been given to collecting data on the
number as well as the character of the micro-organisms present.
The time devoted to this feature of the work was limited and
the data secured is consequently more or less fragmentary, but
it appears of sufficient importance to warrant presentation. The
material examined was secured from several sugar places and
at various intervals during the season.

For the purpose of enumeration the ordinary poured plate
method with dilutions was employed. Two types of media were
used. The first was ordinary nutrient agar ; the other, a synthetic
agar, less well adapted to the cultivation of bacteria than is the
ordinary agar but suited to the development of yeasts and molds.

It had the following composition :

Water 1000 parts

Dextrose 100.

Peptone 20.

Ammonium nitrate 2.5

Magnesium sulphate 5.

Potassium nitrate 2.5

Potassium phosphate 2.5

Calcium chlorid 0.1

Agar 15.0 "

It should be understood that the figures given in the tables

immediately succeeding are only approximate and that in most

cases they are probably below the maximum because of the fact

that the dilutions employed were usually insufficient to prevent

serious crowding on the plates. The blank spaces in the column

headed "synthetic agar'' may be taken as an indication that the

sap in question was plated only on nutrient agar.

Micro-organisms of Maple Sap

339

NUMBER OF ORGANISMS IN SOUS MM'l.i. SAP
The results obtained with various samples of sour sap plated
at one or another time during the progress of the work are
given below in tabular form. Some of the material plated was
just beginning- to show signs of clouding while in other cases the
process was well advanced. In general the degree of decom-
position and the number of organisms found bore a direct re-
lation to each other.

As may be seen from the table the count obtained on nutrient
agar varied from 320,000 to 141,420,000 per cc. Allowing an
average size of 1 micron by 3 microns the organisms represent
approximately one-third the volume of the most heavily infected
sap.

TABLE 1. ORGANISMS PER CC. IN SOUR MAPLE SAP

NO.1
1

2

O
O

4

5

6

7

8

9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
23
2G

Nutrient
agar

3,084,000
0,100,000

21,300,000

6,300,000

1,272,000

2,785,000

320,000

2,000,000

843,000

975,000

8,276,000

1,950,000

6,500,000

19,092,000
1,357,000
1,909,000
1,751,000

10,000,000

88,200,000
1,950,000

14,500,000
8,850,000
7,300,000
2,150,000
5,540,000
7,500,000

Synthetic
agar

200,000
2,000

13,000

715, 000

30,000
200,000
175,000

20,000

1.300

500,000

3,000

254,500

2,450,000

10,000

500
30,000
17,500

No.

27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52

Nutrient
agar

14,000,000

5,667,000

4,225,000

19,500,000

23,000,000

9,750,000

325,000

4,875,000

10,400,000

43,875,00(1

11,275.01)0

56,250,000

2,250,000

1,625,000

12,350,000

3,262,500

23,400,000

29,250,000

11,700,000

9,480,000

14,670,000

6,500,000

73,125,000

65,000,000

13,000,000

141,420,000

Synthetic
agar

9,546,000
51,000

15,000

37.00(1

10,500

407.000

60,000

65.000

646,300

ST, 75(), Oilii

100,000

3. 040. 000

•This series, 1-52, is entirely distinct from the similarly numbered
series later discussed on pages 351 to 365.

341 1

Bulletin 167

NUMBER OL ORGANISMS IN SWEET MAPLE SAP

Jn contrast to the figures in the preceding tahle the results
obtained from plating several samples of normal sap are of in-
terest. In some cases the sap to be analysed was gathered with
unusual care in order to secure as light an infection as might be
without special apparatus, while in other cases it was taken
from the commercial supply and is representative of the ordinary
sap of the orchard in which the work was done. These two
classes of material are grouped in separate columns. The higher
figures in the column headed "commercial sap'' were obtained
late in the season from material which was developing an un-
pleasant flavor and which clouded promptly when placed in
st< >rage for a few hours. In one of these cases (11) the count
obtained is higher than that from many of the sour saps reported
above, and in the other four (4, 5, 10, 12) it is very high.

TABLE 2. ORGANISMS PER CC. IN SWEET MAPLE SAP

No.

Carefully collected sap
Nutrient Synthetic

agar

agar

1

120

140

2

KM)

45

::

500

160

4

5

0

5

30

s

<;

66

17

7

59

7

No.

Commercial sap

Nutrient Synthetic

agar agar

1

1,300

16

2

::,700

1,000

::

3,800

700

4

:::>.:>oo

10.000

5

162,000

325,000

t;

900

610

7

1.000

500

8

140

140

9

220

220

10

66,600

44,000

11

1,000,000

1,100

12

200,000

13

2,600

2,700

14

9,290

7S4

The influence of cleanliness upon the number of organisms
developing in maple sap may be seen in the results obtained in
the following experiments.

Several trees were tapped with care, precautions being taken
to prevent the introduction of inert matter carrying micro-

M [CRO-ORG W ISMS OF M Al'l.i-; S KT

341

organisms into the tap-hole. Sterile metal spouts and buckets were
used and the covers employed were fastened to prevent blowing

in the wind. The sap was gathered daily, and after each col-
lection the buckets were thoroughly scalded in order to main-
tain practical sterility. The plates poured from sap obtained on
March 26 and April 6 appear in table 3.

TABLE 3. ORGANISMS PEE CC. 1 N SAT OBTAINED l\ STERILIZED CONTAIN! BS

Nutri-

Syn-

Nutri-

Syn-

ent

thetic

ent

i aetic

Date

Tree

agar

agar

Date

Tree

agar

agar

3/26/11

A

3

4/6/11

A

li

10

If

B

6

a

B

4

2

(t

C

250

a

C

3

G

It

D

0

•«

D

4

2

E

30

it

E

0

5

a

F

2

tt

F

2

32

BACTERIAL. CONTENT OF SAP OBTAINED UNDER SEPTIC CONDITIONS
AS COMPARED WITH THAT OBTAINED UNDER ASEPTIC CONDITIONS

Trees which were running sour sap were retapped about 4
inches to one side of the old tap-hole. Sterile spouts and buckets
were hung at the fresh wounds, while the sour spouts and buckets
were allowed to remain undisturbed except that the sap was
emptied from the old bucket at the time the clean one was hung
at the fresh tap-hole. The sap was allowed to run from each
spout for a few hours, after which samples were collected and
plated. The results are tabulated below in table 4.

TABLE 4. ORGANISMS PER CC. IX SAP FROM FRESH AND FROM SOUR TAPHOL1 S

Fresh tap

Sour

tap

Nutrient

Synthetic

Nutrient

Synthetic

Tree

agar

agar

agar

agar

G

5

0

73,125,000

87,750,000

H

8

4,875,000

I

70

9,750,

J

12

3,250,000

K

10

1,:: oo.OOO

34-2

Bulletin 167

INFLUENCE OF TAP-HOLE INFECTION UPON THE FLORA OF SAP

For the purpose of this series of experiments 6 trees were
tapped with unusual precaution to avoid infection in the hole at
the time of tapping. The spouts used were of galvanized iron
and were thoroughly sterilized before using, but remained un-
disturbed and without protection, other than the bucket cover,
during the season. Bright tin buckets provided with covers
were used. They were thoroughly washed and scalded at fre-
quent intervals, usually daily, in order to prevent the accumu-
lation of organisms, which otherwise occurs in the buckets as
the season advances. There was, of course, the usual oppor-
tunity for increasingly heavy contamination of the flowing sap
by organisms developing in the tap-hole and spout. The in-
terval between scalding the bucket and plating the sample varied
somewhat with the temperature but was usually about five hours.
The progress of infection is shown in table 5. It is interesting
to note that most of the colonies developing in the sap from tree
number 3 were of a single species. It seems probable that this
organism was carried into the tap-hole at the time of tapping
and became established there to the practical exclusion of other
species.

TABLE 5. ORGANISMS FROM TAP-HOLE AND SPOUT PFU CC. OF MAPLE SAP

Nutri-

Syn-

Nutri-

Syn-

ent

thetic

ent

thetic

Date

Tree

agar

agar

Date

Tree agar

agar

: 20/10

1

1

4

3/25/10

1

2

2

**

2

4

0

tt

2

4

12

..

3

940

680

tt

3

12600

12600

u

4

2

2

ti

4

2

6

..

5

4

4

H

5

7

3

n

6

50

1

tt

6

11

10

5/22/10

1

2

2

4/4/10

1

1300

910

n

2

40

4

ti

2

400

0

..

3

1170

1040

tt

292500 300000

u

4

6

1

(1

4

1300

0

ti

5

10

tt

5

100

tt

it

C

4

2

a

6

1000

16

The figures given above indicate that in most cases the
infection in tap-hole and spout is slight during the early part ^^

Micro-organism 6i Maple Sap ;i;

the season but that it becomes more serious as the spring ad-
vances. In the case of tree number 3, however, there is a
gradual, constant increase in the infection from the very first.

INFLUENCE OF THE CONTAINER ON THE BACTERIAL FLORA

The influence of the container upon the bacterial flora < f
sap is strikingly illustrated by the following experiment. ( >n
March 22, 1910, the sap which had flowed from the 6 trees men-
tioned in the previous table during parts of March 21 and 22.
was mixed together and plated. The number of organisms per
cc. upon nutrient agar was 120, on synthetic agar, 140. Most
of these were of the type characteristic of tree 3 as noted above
On the morning- of March 23, old but sound wooden buckets
previously used in the orchard were thoroughly washed and
hung at the 6 trees. The sap was collected, mixed and plated on
the afternoon of the next day. The counts on nutrient agar
averaged 6,500,000, and on synthetic agar, 500,000. The temper-
ature and general weather conditions during the four days
covered by this experiment were essentially similar. That this
increase of organisms was due to the buckets employed may
be seen by reference to the bacterial count given in table 5 for
March 25, when clean tin buckets were again employed. While
the evidence goes to show that the initial infection from the tap-
hole of tree 3 was greater on March 23 and 24 than on March
21 and 22, there is no indication that this was the case with the
other trees. It should also be noted that the plates poured
on March 24 contained no large proportion of colonies char-
acteristic of the organism of tree 3, and that the quality of sirup
obtained (number 67 page 272) was n°t characteristic of the
organism in question: hence it would seem that the container
must be regarded as the chief source of the increase. See Plates
VI and VII.

GENERAL PLAN OF FIELD EXPERIMENTS

Having established the fact that large numbers of bacteria
and other micro-organisms are constantly associated with spoiled

344 Bulletin 167

sap, it remained to determine the influence (if these agents upon
the quality of sirup. Several hundred pure cultures of the pre-
dominant organisms occurring in various types of spoiled sap
were isolated and studied more or less critically. After their
character had been roughly determined, certain ones were se-
lected for the inoculation experiments to be described presentlv.

The Orange county sugar orchard in which the field ex-
periments were carried out is situated upon a western slope
but is exposed to north winds. The soil of the greater part of the
place is wet. and the orchard lias had the reputation of producing
a grade of sirup of only medium standard both in flavor and
color. See Plate I.

The g'eneral plan followed during the first season was to
secure sap as free from bacteria as was feasible without steriliza-
tion, and to introduce a sufficiently heavy inoculation of the
specific organism to produce an overgrowth of the introduced
species. In some of the later series pasteurization was employed
before inoculation, and during the last season fractional steriliza-
tion was attempted. Following inoculation, after a period of
incubation, the saps were concentrated to sirups under uniform
conditions. The sirups were placed in glass jars, sterilized,
sealed and shipped to the station bacteriological laboratory at
Burlington, where they were stored in the dark for later scoring
as to color and flavor, and subjected to a complete chemical
analysis. As suggested above, the details of handling the sap
before and during incubation varied somewhat from time to
time. The details of these variations will lie explained with the
discussion of the individual samples.

In addition to the inoculation experiments referred to, which
were usually confined to the early runs of the season, an attempt
was made to procure late run sap relatively free from infection.
in order to compare it with material drawn at the same time
from the same trees under ordinary conditions. For this pur-
pose trees which had begun to run sour were selected and. with-
out disturbing the original tap-hole, spile or bucket, another spout

Micro-organisms ob M \ri.i-: Sap ;,'b~>

was introduced into the tree about 4 inches to one side of the
original wound. Great care was observed to secure for this
tap-hole as entire freedom from infection as was possible with-
out special apparatus, and sterile spouts and buckets were em-
ployed. At the time of retapping, the sap in the sour bucket
was emptied but the receptacle was not washed. In this way
two samples of sap were obtained from the same tree at the
same time, one of which was relatively free from micro-organ-
isms, while the other was heavily infected. These two saps were
concentrated to sirup under practically identical conditions and
submitted to the same subsequent treatment as were the other
>irups. These experiments were intended to show whether the
changes which have been demonstrated to occur in sap near the
close of the season were due entirely to the action of foreign
organisms, or whether thev should be ascribed in part or in en-
tirety to physiological changes within the tree.

METHOD OF SIRUP SCORING

At the close of each season the sirups which had been made
during the preceding weeks were scored for flavor by a com-
mercial expert who was entirely unfamiliar with their history.
A series of numbered beakers of uniform appearance were placed
on a table and each received a few ounces of the sirup correspond-
ing in number to that of the beaker. The expert tasted the
sirups and assigned each to its respective grade. After the
entire series had been scored, and a record made of the results,
the samples were rearranged in such a way as to insure the loss
of their identity, except by the small number on each beaker, and
the judge was asked to regrade them. In cases of special im-
portance the samples were repeatedly disarranged and passed
back to be regraded. Incredible as it may seem they were in-
variably placed in the grades originally assigned them and usually
in the same relative position within the grades. Not a single
instance of contradiction occurred among the entire 12S samples.3

'The Station is under obligation to Mr. Otto Ludwig 0!' Burling-
ton for his invaluable and experl assistance.

346 Bulletin 167

Six grades of flavor were recognized, the last two of which
were reserved for material possessing the characteristic flavor
known to experienced sugar makers as "buddy.'' The term
'"buddy" as employed in this bulletin should be understood to
designate that peculiar flavor which occurs only in sirup made
from late run sap drawn after the buds have begun to open.
Much of the material popularly spoken of as "buddy" does not
possess this distinctive character, which is difficult to describe
but is instantly recognized by one who has become familiar with
it.

Number 1 sirups possess a very high degree of excellency.
X umber 2 sirups are also excellent but are slightly inferior
to number 1.

X umber 3 sirups possess the characteristic maple flavor
but, in addition, there is present an unpleasant taste which de-
tracts from their value.

Number 4 sirups lack maple flavor, possess an unpleasant
foreign taste to a marked degree, and are to be regarded as of
very poor quality.

Number 5 applies to sirups which possess the "buddy" flavor
but are otherwise excellent.

Number 6 sirups combine the "buddy" flavor with the other
foreign flavors previously mentioned.

Sirups of grades 5 and 6 can be marketed only with diffi-
culty, and cannot be added to higher grade sirups even in small
amounts without rendering the entire mixture undesirable for
table purposes. Number 4 sirup does not find a ready market
for domestic use unless mixed with higher grade goods.

The sirups were also graded carefully according to color.
The method employed for color determination is that suggested
by Bryan. The colors consist of a series of twenty standards
for the preparation of which Bryan gives the following direc-
tions.1

•U. S. Dvpt. Agr., Bu. Chem., Bui. 134, p. 15 (1911).

Micro-organisms of Maple Sap

:;ir

"The materials used are (i) pure glycerin and (j) a cara-
mel solution, which is prepared as follows:

Heat 6 grams of pure sugar to 2120 C. for one-half hour
in a flat bottomed aluminum dish and dissolve the caramel
formed in boiling water, evaporate to a small volume, and make
up to 200 cc. with glycerin. The oven for caramelizing the sugar
(fig. 1) is constructed as follows:

A and A' are heavy sheets of asbestos board iS cm. (7
inches) square, A' being perforated near one aV^c by a hole

for a cork supporting the thermometer
d: b is a sheet-iron cylinder 15 cm. (6
inches ) in diameter ; ; c is a tin can 9 cm.
(3/^> inches) in diameter, which is filled
with paraffin to within 1 cm. (l/2 inch)
of the top. This can rests on the pipe-
stem triangle e. The bath or oven is
supported on a tripod and is heated by
two burners. One burner is so ad-
justed as to keep the bath at 2120 C.

Bring the temperature of the oven up
to 2120 C, using both burners. Then re-
Pig, j. Apparatus for prepa- move the asbestos cover carrying the

tion of standard caramel. J °

thermometer and place 6 grams of sugar in a flat-bottomed
aluminum dish 7 cm. (2^4 inches) in diameter and 1.5 cm. (^
inch) deep, and put it in the can containing the paraffin. Replace
the cover at once and as soon as the temperature reaches 2080 C.
turn out one burner and keep the bath at 2120 C. by carefully ad-
justing the other one. At the expiration of thirty minutes from
the time the sugar was placed in the bath, dissolve in boiling wa
ter, and make up as described. The aluminum dish should not be
less than 1.5 cm. (^ inch) deep, since the sugar melts before
caramelizing and runs to one side of the dish, which, if too shal-
low, will tilt, fill with paraffin, and sink.

With the ingredients thus prepared, the scale of colors is
made up by mixing as indicated in the following table :"

ra

348

TABLE G.

Bulletin i6y

AMOUNTS HI INGREDIENTS TO BE USED I .\ PREPARING SOLUTIONS
FOB THE COLOR SCALE

Color number

Caramel solution,

Glycerin,

grams

grams

1

0.00

35.00

2

0.25

34.75

3

0.50

34.50

4

0.75

34.25

5

1.00

34.00

G

1.50

33.50

7

2.50

32.50

8

3.50

31.50

9

1.50

30.50

Hi

5.50

29.50

II

7.0U

28.00

12

8.50

26.50

13

L1.00

24.00

1 1

14.00

21.00

ir.

17.00

18.00

16

20.00

15.00

17

23.50

11.50

18

27.00

8.00

19

31.00

4.00

20

35.00

0.00

The standard colors were placed in screw-capped vials of
perfectly clear glass having the same internal diameter. The
samples to be examined were placed in similar vials and the
colors compared by transmitted light.

For commercial purposes sirups are usually divided into
three grades of color. The point of division between the stand-
ards fluctuates somewhat according to the locality and season.
In a good season and in a locality where light sirups are pro-
duced, numbers up to and including 7 are regarded as first
quality, while number 11 represents the dividing line between
second quality and third. On the other hand in years or in
localities characterized by dark sirup, the first eleven grades of
color may be accepted as first quality, while number 15 becomes
the dividing line between second and third qualities.

Tn addition to the rating for color and flavor a numerical
score card was prepared in which color and flavor were both
considered. In scoring, number 1 was ranked as 100. number 2
as 95, and so on, subtracting 5 from the score for each fall of

M [cro org \.\ tSMS 'ii; Maple S vf

349

i in grade. A number 20 sirup therefore received a value ol
5 and anything darker than jo a value of <>. A table "l" value- 1
given below.

TABLE 7. VALUES ASSIGNED TO COLOR GRADES

Color

Value

1

100

2

95

3

90

4

85

5

SO

6

7.-,

7

70

8

65

!•

60

10

55

Color

Value

11

50

12

45

13

40

14

35

15

30

If.

25

17

l!ii

18

15

19

10

20

5

20+

0

In scoring- for flavor certain samples were found which
clearly were not sufficientlv fine to he ranked as number 2 and
at the same time were superior to number 3. Two fractional
numbers were used to designate these groups. These were 21
and 2-. Similarly one group was introduced between 3 and 4,
which is designated as 3*. In preparing the numerical score
card the following table of values was employed:

TABLE S. VALUES ASSIGNED TO FLAVOR GRADES

Flavor

Value

Flavor

Value

Flavor

Value

1

100

22

80

4

50

2

90

3

75

5

20

21

85

31

G5

6

0

The numerical score was obtained by multiplying by 5 die
sum of the numerical values assigned for color and for flavor.
The highest score theoretically possible is thus seen to he 1.000,
hut as a matter of fact no maple sirup has the color of clear
glycerin so that a sirup of number 1 color is not to be expected,
and the highest attainable score becomes 975.

[noculation Experiments ix 1900

The first inoculations were made in 1909 and were carried
out at a farm house some distance from the sugar woods where

350 Bulletin 167

most of the field studies were conducted. The sap was collected
in clean tin buckets provided with japanned covers. The spouts
used were of galvanized iron. Care was exercised to keep the
buckets clean and sweet in order to avoid an accumulation of
micro-organisms in the sap. The sap was gathered in clean tin
cans such as are usually employed for transporting milk, and
carried to the farm house in which the temporary laboratory
was then established. It was divided into portions of sixteen
quarts each and placed in new tin buckets provided with covers.
Certain portions were reserved as controls and the remainder
were inoculated by adding 70 cc. of a young actively growing
culture of the specific organism selected. The controls were
treated with similar amounts of sterile culture media. The in-
oculated samples were placed on a table and incubated for three
days. The temperature variation during the respective incuba-
tion periods is shown in the accompanying graphs taken from
the tracings made by a self-recording thermometer placed near
the samples. The actual temperature of the sap was of course
much more constant than that of the air. After an incubation
period the various portions were made into sirup, each of the
several saps being evaporated in a bright sugaring-off pan on
a kitchen stove until condensed to a volume of about one quart.
It was then transferred to a white, agate-ware basin, and evap-
orated as rapidly as possible until the proper concentration, as
indicated by a thermometer, was reached. The process from
cold sap to sirup, required about an hour and a half for its
completion, except in the case of one sample when the time
was intentionally prolonged. The samples made during the first
season are those discussed in bulletin 151 as numbers 1 to 26
inclusive. The numbers used at that time, however, have not
been retained in the present publication. Moreover the method
of scoring employed in this article is not the one used in the
former issue, a fact which explains any apparent lack of uni-
formity. The numbers used in the present bulletin and in the
former one are given below in tabular form :

Micro-organisms of Maple Sap

351

New number

Old

number

New

number

Old number

(Bui. 167)

(E

tul. 151 )

(Bui. L67)

( Bui. L51)

1

1

11

18

2

7

L5

20

::

8

L6

L9

4

9

17

21

5

10

IN

22

6

2

19

23

7

12

20

5

8

11

21

6

9

13

22

24

10

14

23

26

11

16

24

3

12

15

25

25

13

17

26

4

Since it was deemed important to conduct the inoculation
experiments with sap from the first runs only, about ioo trees
were tapped for experimental purposes as early as it seemed
probable that any sap could be obtained.

DETAILED DISCUSSION OP SAMPLES

SERIES I : SIRUPS I TO 6

The sirups of this series were made from the first run of
the season which occurred on March 24, 1909. It remained in
the woods over night and was brought to the field laboraton
the following- morning- and divided into six portions. Four <>i
these were inoculated and two retained as controls. They were
held for three days, the controls being kept at a temperature of
from o° to +io° C. The incubation temperature of the inocu-
lated samples is indicated in the temperature graph (fig. 2).

J 3C J? Jrf* 33 9Z *5 ■«/ J

S4 S? C

0 0 i

«. 4» 7*. 7S 7

t ti « tr >• »/ 9i : 3 1

U-

-1

'{

r, **t 1

J <

u;

n

Si-

80

fS

/

V

IS

\

A

1

10

)

I

>

s

N

',

>

es

7

i

1

v

so

y — 1
\

V

\

-\j

f

■■

1

Fig. 2. Graph of incubation temperatures for series 1 ; saps 1 to G In-
clusive. The arrow heads on next to the bottom line from left to right indi
cute respectively the time of inoculation, the time of evaporation of the first
sample and the time of evaporation of the last sample.

852 Bulletin 167

The inoculated saps promptly developed a cloudy appearance
and showed all the characteristics of so-called sour sap of the
late runs. (See Plate V). The details of the various samples
follow :

i. Number 1 was a control used to determine the influ-
ence of long boiling- upon the quality of the product. The sap
was held between three and four days at a temperature only
slightly above freezing. At the end of that time it appeared to
be as fresh and sweet as when brought from the woods. It was
evaporated to sirup over so slow a fire that six hours were re-
quired to concentrate the 16 quarts of sap which constituted the
sample. Calculated to a dry matter basis, the sirup contained
96.84$ sucrose and 1.07$ invert sugar. The color was 7, flavor
1, and grade 850; depreciation from control. (No. 6) color 1.
flavor o, and score 25.

2. Number 2 was inoculated with fluorescent organism
XXXIIP. It developed a cloudy appearance with the greenish
cast characteristic of the so-called g"reen sap. The sirup con-
tained 96.79$ sucrose and 1-55% invert sugar. The color was
14. flavor 4, and score 425; depreciation from control, color 8,
flavor 3, and score 450.

3. Number 3 was inoculated with fluorescent organism
XXXVI. It developed the characteristic green type of sour-
ing. The sirup contained 96.73% sucrose and 2.09$ invert
sugar. The color was 11, flavor 2, and score 700; depreciation
from control, color 5. flavor 1, and score 175.

4. Number 4 was inoculated with fluorescent organism
L, and developed a green type of souring. The sirup contained
96.54$ sucrose and 1.56% invert sugar. Tbe color was 9,
flavor 2, and score 750; depreciation from control, color 3, flavor
1 , and score 125.

5. Number 5 was inoculated with fluorescent organism
LIII. It developed typical green souring. Tbe sirup contained

1 See Part III c. pages 521 to 599 for a description of the members
11! the fluorescent group employed in this and subsequent samples, finni-
maries of statistical data occur on pages 550-551, 598-599,

Micro-organisms of Maple Sap

353

97.169? sucrose and 1.44 °/t invert sugar. The color was 11,
flavor 2, and score 700; depreciation from control, color 5, flavor
I, and score 175.

0. Number 6 was a control having exactly tbe same history
as number 1, except that it was concentrated over a brisk fire
under conditions which required less than 2 hours to reduce it
to sirup. The sirup contained 95.41% sucrose and 097% in-
vert sugar. The color was 6, flavor 1, and score 875.

SERIES 2: SIRUPS 7 TO 12

Following the run of March 24 no more sap was obtained
until March 27, on which date a light run occurred and enough
sap was secured for seven samples. Six of these were inocu-
lated and handled under conditions similar to those described for
series 1. The air temperature maintained during the incubation
may be seen by referring to the graph (fig. 3). The seventh

CO
Si

■IS
10

r

-

? 1

S 11 14 21 ■

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Fig. .'5. Graph of incubation temperatures for series 2 ; saps 7 to 12 in-
clusive. The arrow heads on next to the bottom line from left to right indi-
cate respectively the time of inoculation, the time of evaporation of the firs'
sample and the time of evaporation of the last sample.

SI

sample was reserved for a control, but, unfortunately, the glass
jar containing the sirup was broken during sterilization and the
sample was lost. From the notes made at the time of boiling
it seems certain that the sirup was at least equal in quality to
number 6 in the preceding series and probably slightly superior
to it. The sirups of series 2 have therefore been referred to
number 6 as a control. One of the saps of this series was titrated
in cold solution against phenolphthalein with N/Too sodium
hydroxid just before evaporation. The reaction is stated in per-
cent of hundredth normal acid.

354 Bulletin 167

7. Number 7 was inoculated with fluorescent organism
L\ I. It developed typical green souring. The sirup contained
96.16% sucrose and 1.64% invert -sugar. The color was 10,
flavor 2, and score 750; depreciation from control, color 4, flavor

1, and score 150.

8. Number 8 was inoculated with fluorescent organism 5.
A typical green sour sap developed. The sirup contained 97.14' <
sucrose and 1.15$ invert sugar. The color was 10, flavor 2,
and score 725; depreciation from control, color 4, flavor 1, and
score 150.

9. Number 9 was inoculated with non-fluorescent organism
XNVf. It developed a milk}' type of souring. The sirup con-
tained 95.61% sucrose and 0.78% invert sugar. The color was
5, flavor 2, and score 850. The color was 1 point higher than
the control, with a depreciation in flavor of I, and in total score
of 25.

to. Number 10 was inoculated with non-fluorescent organ-
ism XXX. It developed a very pronounced milky type of sour-
ing, accompanied by an unpleasant odor. The sirup contained
95.80', sucrose and o.j~'< invert sugar. The color was 6.
flavor 3, and score 750; depreciation from control, color o, flavor

2, and score 125.

ir. Number it was inoculated with Bacillus acrris, strain
LXXXYTI, obtained from stringy sap. The sample developed
a deep milky color and a yeasty odor and became stringy. The
reaction was 170% N/100 acid. The vapor evolved during the
concentration was extremely unpleasant, and was sufficiently
noticeable to attract the attention of a sugar maker who was
passing the house where the field laboratory was established.
Although he was entirely unaware of the nature of the experi-
ments, the steam borne to him through an open window elicited
ilk' remark, "It smells like the last of sugaring.'* The sirup con-
tained 96.12% sucrose and 1.72', invert sugar. The color was
7, flavor 4, and score 600; depreciation from control, color 1.
flavor 3, and score 275.

Micro-organisms oi? Maple Sap

.;.,.,

[2. Number 12 was inoculated with non-fluorescen! organ
ism I, obtained from stringy peas. A milky type of souring
developed. The sirup contained 95.71^ sucrose and 2.23$
invert sugar. The color was 6, flavor 3. and score 750; depreci-
ation from control, color o, flavor 2, and score 125.

SERIES 3: sirups 13 To 20

The sap for this series was secured April 1 and handled
in the manner described for the two preceding series. The
temperature of incubation may be seen by referring to the
graph (fig. 4).

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1

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Fig. 4. Graph of incubation temperatures for series 8 ; saps 13 to 20 in
elusive. The arrow heads on next to the bottom line from left to right indi
cate respectively the time of inoculation, the time of evaporation of the first
sample and the time of evaporation of the last sample.

13. Number 13 was inoculated with gray yeast 24. A mix-
ture of organisms developed. Apparently the fluorescent bac-
teria naturally present in the sap were not held in check by the
yeasts, and the product seemed to be the results of the combined
action of yeasts and bacteria. The sirup contained 93.34' <
sucrose and 2.53% invert sugar. The color was 7. flavor 2. and
score 800; depreciation from control, color 1, flavor 1, and
score 75.

14. Number 14 was inoculated with red yeast 25. A mix-
ture of organisms developed as was the case with the previous
sample. The reaction was 9% N/100 acid. The sirup con-
tained 95.30% sucrose and 3.01% invert sugar. The color
was 10, flavor 2, and score 725; depreciation from control, color
I, flavor 1, and score 150.

356 I'.II.I.KTIN 167

15. Number 15 was inoculated with red yeast LXII and
incubated for 3 days. As with other samples previously reported
where yeasts were employed a mixture of organisms developed.
The reaction was 5% N/100 acid. The sirup contained 93.2!
sucrose and 2.98/r invert sugar. The color was 8, flavor 2.
and score 775; depreciation from control, color 2, flavor 1,
and score 100.

16. Number 16 was inoculated with green mold LXIY. The
bacteria naturally present developed in conjunction with the
mold and the sample had the appearance characteristic of the
green type of souring, but it was evident from the tufts of
mycelium present that the mold had made good growth. The
reaction was 8.2$ N/100 acid. The sirup contained 95.73$
sucrose and 2.61 '/< invert sugar. The color was 10, flavor 4.
and score 525; depreciation from control, color 4. flavor 3. and
score 350.

17. Number 17 was inoculated with green mold LXV.
A mixed infection resulted. The reaction was 8% X Too acid.
The sirup contained 92.76% sucrose and 3-47% invert sugar.
The color was 10, flavor 2, and score 725; depreciation from
control, color 4, flavor 1, and score 150.

18. Number 18 was inoculated with green mold LXXIX.
A mixed infection resulted. The reaction was \2('< X 100 acid.
The sirup contained 91.65% sucrose and 3.12% invert sugar.
The color was 10, flavor 2, and score 725 ; depreciation from
control, color 4, flavor 1, and score 150.

19. Number 19 was inoculated with a composite culture
consisting of fluorescent bacteria, yeasts and molds. The re-
action was 5% N/100 acid. The sirup contained 93.28% sucrose
and 4.09% invert sugar. The color was 9, flavor 3. and score
^7$: depreciation from control, color 3, flavor 2, and score 200.

20. XTunber 20 was held without inoculation at low temper-
ature during the incubation period and reserved as a control
The reaction was 2% N/100 acid. The sirup contained 95.37

Micro-organisms oi? M.\ri.r Sac 357

sucrose and 1.58% invert sugar. The color was 6, flavor 1,

and score 875.

SIRUPS PROM LAST RUN SAP IN I909

No more inoculation work was conducted during the season
of 1909, but several samples of sirup were made with a view
of determining- the possible influence of physiological changes
in the tree upon the quality of sirup. As previously stated,
trees were selected for this purpose which were "running sour,"
that is to say trees from which clear sap was no longer being
obtained. The spouts usually "dry up" or cease to run quite
promptly after the appearance of this phenomenon. The aim
was to wait till the last day a tree was likely to run before making
the experiment. A fresh tap from which the sap flowed clear
was then made about 4 inches to one side of the old one. The
two types of sap thus secured from the same tree at the same
time would presumably be very similar in character until they
left the vascular tissues. That from the old taphole, however,
becomes very heavily infected as it flows from the spout while
the other is relatively free from infection. Any difference in
quality of the two sirups obtained under such circumstances
would seem to be attributable to the influence of micro-organ-
isms, while any differences in quality between controls of the
first runs, and the last run material from the fresh taps, would
reasonably be ascribed to physiological changes which had taken
place within the tree.

21. Number 21 was made from sweet sap obtained by re-
tapping the first sour tree observed during the season. This
was drawn on April 12. The sirup contained 95.19% sucrose
and 0.82% invert sugar. The color was 3, flavor 1, and score
950. The color was 3 grades superior to the first run control
of the season, the flavor equal, and the total score 75 points
higher.

22. Number 22 was made from sour sap obtained from the
same tree at the same time as the sap for 21. The sap was

358 JJuuxnN 167

only slightly cloudy and had all run the day it was concentrated.
The sirup contained 95.27% sucrose and 2.76% invert sugar.
The color was 8, flavor 3, and score 700; depreciation from con-
trol (number 21), color 5, flavor 2, and score 250. Deprecia-
tion from first run control, color 2, flavor 2, and score 175.

23. Number 23 was made from sour sap obtained April
12 and held 24 hours. The sirup contained 92.56% sucrose
and 3-55% invert sugar. The color was 14, flavor 3, and score
550; depreciation from control (number 24), color 7, flavor 2.
and score 300. Depreciation from first run control, color 8,
flavor 2, and score 325.

24. Number 24 was made from sap obtained at the same
time and from the same tree as that for number 23. It was
drawn from a fresh tap, but was held for twenty-four hours
before concentration. The sirup contained 96.02%' sucrose and
0.61% invert sugar. The color was 7, flavor 1. and score 850.
Depreciation from first run control, color I, flavor o, and score

25.

25. Number 25 was made from sour sap which flowed

April 16. The sirup contained 94.05' r sucrose and 3.13'-
invert sugar. The color was 14, flavor 3, and score 550: de-
preciation from control (number 26), color 9, flavor 2, and score
350. Depreciation from first run control, color 8, flavor 2, and
score 325.

26. Number 26 was obtained from the same tree as number
25 and at the same time, but from a fresh tap-hole. The samples
were concentrated the same day they flowed. The sirup con-
tained 96.42% sucrose and 0.5 \' < invert sugar. The color was
5, flavor 1, and score 900. The color was one grade superior to
the first run control, the flavor equal, and the total score 2^
points higher.

The results of this single season's experiments taken alone
would seem to indicate that the color and flavor of sirup are
not impaired by physiological changes in the tree occurring dur-
ing the sugar season. It is important to note, however, that

Micro-organisms or Maplb Sap •"■•"''.,

the season of 1909 was peculiar. There were no warm periods
of importance and the interval between the first run and the
last was only three weeks. The commercial season was even
shorter since the experimental trees were tapped before the real
season opened and most sugar makers in the vicinity gathered
their buckets before April 16.

Inoculation Experiments in iqio

The experience gained in 1909 emphasized the importance of
more carefully controlled inoculation experiments. To facili-
tate the progress of the studies it seemed best to establish a lab-
oratory where the more simple bacteriological technique could
be carried out in close proximity with the woods. Accordingly,
before the opening of the sugar season in 1910, a small rough
wooden building was put up in the sugar woods for this pur-
pose. (See Plate II). The floor space available was about 6"
by 24 ft. The equipment consisted of an old fashioned box-
stove, the top of which could be easily removed to be replaced
by the evaporating pan, a blue-flame oil stove with an oven, an
Arnold steam sterilizer, a microscope, Petri dishes, test tubes
and other glassware, a few chemicals and the necessary sugar
tools. (See Plate III). Since the laboratory was of rough
construction and consequently became cold quickly, it was nee
sary to provide some apparatus in which the inoculated sap
could be stored, in order to maintain a reasonably constant tem-
perature during the period of incubation. For this purpose
a rather crude modification of the modern fireless cooker was
provided. (See Plates III and IV). This consisted of a box
the approximate interior dimensions of which were, height 20
inches, width 40 inches, and length 16 feet. The walls were
made of 2 double layers of matched sheathing between which
was an air space of I inch. Building paper was used between
each of the double lavers of sheathing- and on each side of the
one inch air space. The top, bottom, and sides were of similar
construction, the cover being so arranged as to break joints .1

360 Buu,etin 167

is customary with the doors of safes or refrigerators. The
cover was formed in sections so as to permit opening one end
without admitting cold air to the entire contents.

It was the first intention to maintain the temperature by
introducing hot freestones into the incubator when the tempera-
ture of the sap began to fall. It soon appeared, however, dur-
ing the extremely cold weather encountered shortly after start-
ing the first series of the year, that this means of control was
inadequate and the more satisfactory method of introducing
buckets of boiling water was resorted to. This raised the air
temperature in the incubator very promptly and the heat was
gradually imparted to the sap. The temperature of the samples
was occasionally tested by introducing a sterile thermometer
into the buckets, and whenever necessary additional heat was
supplied.

A self recording thermometer was placed in the incubator
from the tracing's of which the temperature curves here repro-
duced were obtained. It should be borne in mind, however,
that these curves by no means represent the temperature of the
sap which was far more constant than that of the air. It never
fell as low nor did it rise as high. The graphs are of value, how-
ever, in that they give some indication of the conditions of in-
cubation.

The material was incubated in new tin buckets which were
thoroughly scalded immediately before filling. They had a
capacity of 20 quarts and were provided with covers. Inocula-
tion was made with a 70 cc. young culture of the specific organ-
ism employed. The controls were treated with an equivalent
amount of the sterile medium which consisted of 65 cc. of sap
and 5 cc. of nutrient bouillon.

Before being made into sirup each sap was thoroughly mixed
and a small sample was withdrawn and sterilized to be preserved
for the purposes of another investigation, the results of which it
is expected will eventually be published by the station chemist.
As a rule the reaction was determined by titrating aefainst

M u mi oroanisms 01' M \ri.i-: Sap ;',;l

phenolphthalein in the cold with N'/ioo sodium hydroxid, and
a bacteriological analysis was made in order to determine the

degree of success which had been attained in inoculation. The
sap was then placed in a thoroughly clean pan over a brisk,
(laming wood fire and evaporated to a volume of about I quart,
when it was transferred to a white agate ware basin and concen-
trated to a density of n pounds to the gallon as indicated by the
thermometer. It was the custom to determine daily the tempera-
ture at which water boiled in the laboratory, and to subtract
from 219 the difference between 212 and the boiling point found,
in order to arrive at the temperature at which evaporation should
be discontinued during that day. Provision was thus made for
fluctuations in the boiling point due to atmospheric conditions.

The sugar season of 1910 opened unusually early, before
the laboratory was quite ready for use. The first run occurred
on March 3 and 4, but the experimental trees were not tapped
until March 6.

series 4: sirups 27 to 40

The sap for this series was collected March 7, and placed
in the incubating buckets in 16 quart portions. It was not so
thoroughly mixed as to insure absolutely uniform chemical com-
position, but was handled in a manner to secure approximate
uniformity. The temperature of all except two of the samples
was raised to 400 C. before placing them in the incubator. One
of these was allowed to stand outside where a temperature of
o° to io° C. prevailed, while the other was heated to 8o° C.
and placed in the incubator. These two samples, together with
one of those heated at 400 C, constituted the controls. When the
temperature of the sap had dropped to 280 C. the samples were
inoculated with their respective organisms and incubated for 3
days. Evaporation of the samples began at 9 a. m., March II,
under conditions which made it possible to average the produc-
tion of a sirup in an hour and a quarter. The temperature of
incubator mav be seen bv reference to figure 5.

362

Bulletin 167

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Fig. 5. Graph of incubation temperatures for series 4 ; saps 27 to 40 in-
clusive. The arrow hearts on next to the bottom line from left to right indi-
cate respectively the time of inoculation, the time of evaporation of the first
sample and the time of evaporation of the last sample.

2"j. Number 2j was the control kept outside the incubator
at a temperature only slightly above its freezing point. Not-
withstanding the low temperature it contained 10,000 organisms
per cc. although there was no evidence of clouding at the time of
evaporation. The sirup contained 96.19% sucrose and 0.30%
invert sugar. The color was 4. flavor 2, and score 875.

28. Number 28 was inoculated with gray yeast 24. The
sap became cloudy, at first greenish, then milky. The organism
was recovered in mixed culture. The plates showed 437,000
colonies per cc. The sirup contained 82.83% sucrose and
12.15% invert sugar. The color was 11, flavor 4, and score 500;
depreciation from control, color 7, flavor 2, and score 375.

29. Number 29 was inoculated with fluorescent organism
CXXXIX. A green type of souring developed, the sap becom-
ing cloudy within twelve hours. The organism was recovered
in practically pure culture, the plates showing 12,330,000 per cc.
The sirup contained 94.19% sucrose and 0.38% invert sugar. The
color was 8, flavor 21, and score 750; depreciation from control,
color 4. flavor o1, and score 125.

30. Number 30 was inoculated with fluorescent organism
CLIX. It was recovered apparently in pure culture from the
green sour sap which resulted. The plates showed 3,262,000
colonies per cc. The sirup contained 93.75% sucrose and
0.89% invert sugar. The color was 8, flavor 2. and score y~'? :
depreciation from control, color 4, flavor o, and score 100.

Micro-organisms of Maple Sap 303

31. Number 31 was inoculated with gray yeast CXXI.
The organism was not recovered, hut the plates showed a count
of 5,667,000 colonics per cc. in which those of the fluorescent
group greatly predominated. The sirup contained 96.62% suc-
rose and 0.30' < invert sugar. The color was 6, flavor 2 and
score 825 ; depreciation from control, color 2, flavor o, and
score 50.

32. Number 32 was inoculated with gray yeast CLXXX.
The organism was not recovered. The plates showed a count
of 5,600,000 colonies per cc, most of which were of the green
fluorescent type. The sirup contained 95.91% sucrose and
0.46% invert sugar. The color was 6, flavor 2, and score 825 ;
depreciation from control, color 2, flavor 0, and score 50.

33. Number 33 was inoculated with gray yeast CLXVIII.
The organism was not recovered. The plates showed 1,950,000
colonies per cc. most of them characteristic of the fluorescent
organisms. The sirup contained 95.61% sucrose and 0.23%
invert sugar. The color was 7, flavor 2, and score 800, depre-
ciation from control, color 3, flavor o, and score 75.

34. Number 34 was the control previously mentioned as
having been heated to 400 C, and placed in the incubator. As
would be expected the sap promptly clouded, developing a green
type of souring. The plates showed a count of 7,312,000 or-
ganisms per cc. Fluorescence was pronounced. The sirup con-
tained 95.86% sucrose and 0.42% invert sugar. The color was 6,
flavor 21, and score 800; depreciation from cold control, color 2,
flavor o1, and score 75.

35. Number 35 was inoculated with red yeast CXI. Deep
clouding developed and the organism was recovered in associa-
tion with large numbers of fluorescent bacteria. The plates
showed a count of 2,255,000 organisms per cc. The sirup con-
tained 95.13% sucrose and 1.15% invert sugar. The color was
12, flavor 21, and score 650; depreciation from control, color 8,
flavor o1, and score 225.

8f)4 Bulletin 167

36. Number 36 was inoculated with red yeast CII. The
sap clouded promptly but only a very few colonies of the organ-
ism were recovered. The plates showed a count of 7,312,000 in
which the fluorescent group predominated. The sirup contained
96.16% sucrose and 0.61% invert sugar. The color was 9,
flavor 21, and score 725; depreciation from control, color 5,
flavor o1, and score 150.

37. Number 37 was inoculated with red yeast CLXXV.
The organism w-as not recovered. The plates gave a count of
8,825,000 per cc. with pronounced fluorescence. The sirup con-
tained 95.88% sucrose and 0.64% invert sugar. The color was
11, flavor 21, and score 675; depreciation from control, color 7,
flavor o1, and score 200.

38. Number 38 was the control placed in the incubator af-
ter heating to 8o° C. The plates showed a count of 162,500
colonies per cc. of which very few developed fluorescence. The
sirup contained 94.80% sucrose and 0.48% invert sugar. The
color was 5, flavor 21, and score 825 ; depreciation from cold
control, color 1, flavor o1, and score 50.

39. Number 39 was inoculated with gray yeast C. Green
souring developed and the organism was not recovered. The
plates showed a count of 9,750,000 colonies per cc. with pro-
nounced fluorescence. The sirup contained 96.18% sucrose and
0.26% invert sugar. The color was 7, flavor 2, and score 800;
depreciation from control, color 3, flavor o, and score 75.

40. Number 40 was a control placed in the incubator with-
out heating. Plates showed a count of 5,850,000 organisms with
the fluorescent group in predominance. The sirup contained
95.05% sucrose and 0.64%^ invert sugar. The color was 9, flavor
2, and score 750; depreciation from cold control, color 5, flavor
o, and score 125.

The results of this series confirmed the suspicion entertained
from the inoculation trials of the previous season's work, that the
yeasts did not develop readily in maple sap, but that the bac-
teria normally present gained the ascendency. The changes in

Micro-organisms of Maple Sap

::<;:,

the sirups of this scries, with the exception of number 28, seemed
to have been very largely produced by the tluorescent group of
organisms. Even the cold control was apparently considerably
injured both in color and flavor by the organisms developing in it
during the incubation period, so that the depreciation in quality
due to their action is probably greater than is indicated by the
figures given above.

That this supposition is well grounded is shown by the re-
sults obtained in later series.

SERIES 5 : SIRUPS 41 TO 48

Following the run obtained on March 7, there was a freeze
and no considerable amount of sap was obtained again until
March 14. The sap was gathered with a team and a sufficient
quantity for 8 samples was withdrawn as soon as it reached the
sugar house. Six of these were heated to boiling and placed in
the incubating buckets while two were left unheated, their tem-
perature being very near o° C. On the morning of March 15,
five of the heated samples were inoculated and allowed to in-
cubate for 3 days at temperatures indicated in the graph (figure

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Fig. 6. Graph of incubation temperatures for series 5 ; saps 41 to 48 in-
clusiye. The arrow heads on next to the bottom line from left to right indi
cate respectively the time of inoculation, the time of evaporation of the lirsi
sample and the time of evaporation of the last sample.

6). Before evaporation the samples were titrated, plated, and
sampled as described for the previous series.

41 Number 41 was a control left standing over night be-
fore being made into sirup on the morning of March 15 when
it contained 66 organisms per cc. and reacted i% N/100 acid.

Sfifi Bulletin 167

The sirup contained 97.82% sucrose and 0.45% invert sugar.
The color was 4, flavor 1, and score 925.

42. Number 42 was inoculated with red yeast CVII. A
milky appearance developed and the organism was recovered in
considerable quantity in association with bacteria producing non-
llnorescent colonies. The plates gave a count of 13,650,000
colonies per cc. The reaction was 19% N/100 acid. The sirup
contained 93.27% sucrose, and 2.14% invert sugar. The color
was 5, flavor 2-, and score 800; depreciation from control, color

1, flavor i2, and score 125.

43. Number 43 was a control heated to boiling and incu-
bated for 3 days. The material became cloudy the third day
and the plates showed a development of 1,625.000 organisms per
cc, mostly of the non-fluorescent type. The reaction was 6.5%
N/100 acid. The sirup contained 94.19% sucrose and 2.64' ',
invert sugar. The color was 7, flavor 2, and score 800; deprecia-
tion from cold control, color 3, flavor 1, and score 125.

44. Number 44 was inoculated with fluorescent organism
CXLVIII. A greenish type of clouding developed and the or-
ganism was recovered in numbers amounting to 9,000,000 per
cc. The reaction was 10% N/100 acid. The sirup contained
93.039? sucrose and 1.40% invert sugar. The color was 9,
flavor 3, and score 675; depreciation from control, color 5, flavor

2, and score 250.

45. Number 45 was a control incubated for 3 days in the
cold. The sap remained perfectly clear. The plates revealed a
bacterial content of 195,000 per cc. The reaction was 4.5%
N 100 acid. The sirup contained 96.28%: sucrose and 0.51%
invert sugar. The color was 4. flavor x. and score 925; depre-
ciation from control (number 4O, color o. flavor o, and score o.

• 46. Number 46 was inoculated with pink coccus (TV.
Pronounced clouding of the milky type and a yeasty odor de-
veloped. The plates showed a count of 14,300,000 organisms
per cc. among which the introduced organism was predomin-
ant. The reaction was 10% N/100 acid. The sirup contained

Mil kiih irg \.\ [SMS "!■' M M'i i. S \r

367

95.07^? sucrose and 1.05$ invert sugar. The color was 6, flavoi
2, and score 825; depreciation from control, color 2, flavor 1,
and score 100.

47. Number 47 was inoculated with pink coccus IV. \
milky type of souring developed. The plates showed a count
of 1,950,000 organisms per cc, the majority of which apparently
were produced by the introduced organism. The reaction was
i).')'. N/100 acid. The sirup contained 88.81% sucrose and
7.90% invert sugar. The color was 6, flavor 3, and score 750;
depreciation from control, color 2, flavor 2, and score 175.

48. Number 48 was inoculated with gray yeast CLXXX.
The sap became milky and developed a yeasty odor. The organ-
ism was recovered in association with bacteria the colonies
of which resembled those of the subtilis group. The plates gave
a total count of 1,625,000 per cc. The reaction was 1(1.7'-
N/100 acid. The sirup contained 93.89'// sucrose and 2.35^
invert sugar. The color was 6, flavor 22, and score 775 ; depre-
ciation from control, color 2. flavor I2, and score 150.

series 6: sirups 49 To 57
Series 6 was made from sap which flowed on March jo. A
sufficient quantity was gathered at four o'clock for 9 samples
and the material was thoroughly mixed to insure uniformity of
chemical composition. Two of the samples were left untreated
while the others were heated to boiling, cooled, and, with the ex-
ception of one, inoculated. The air temperature maintained dur-
ing the 3 days' incubation is shown in the accompanying graph
(figure 7).

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Fig. 7. Graph of incubation temperatures Eor series 6 ; saps !'.» to 57 in
elusive. The arrow heads on next to the bottom line from left to right indi-
cate respectively the time of inoculation, the time of evaporation of the first
sample and the time of evaporation of the last sample.

868 Bulletin 167

49. Number 49 was a control evaporated immediately after
gathering. A bacteriological analysis showed a count of only 100
organisms per cc. The reaction was 1% N/100 acid. The sirup
contained 96.51% sucrose and 0.42% invert sugar. The color
was 2, flavor 1, and score 975. This sirup was considered the
best specimen obtained during the entire period covered by the
work.

50. Number 50 was inoculated with pink coccus XLIX.
Deep clouding resulted and the coccus was recovered in prac-
tically pure culture. The plates showed a count of 1,950,000
organisms per cc. The reaction was 8% N/100 acid. The sirup
contained 93.83% sucrose and 2.20' '< invert sugar. The color
was 6, flavor 21, and score 800; depreciation from control, color
4, flavor i1 and score 175.

51. Number 51 was inoculated with pink coccus CVI. The
sap became deeply clouded. The organism was recovered, the
plates showing a count of 4,225,000 per cc. with the introduced
species in predominance. The reaction was 3.5% N/100 acid.
The sirup contained 97.30% sucrose and 0.65% invert sugar.
The color was 4, flavor 2, and score 875 ; depreciation from con-
trol, color 2, flavor 1, and score 100.

52. Number 52 was inoculated with gray yeast CLXII.
A milky white clouding developed. The organism was recovered
in mixed culture. The plates showed a count of 1,275,000
colonies per cc. About 30,000 of these were characteristic of
the introduced organism, while most of the others were bac-
terial colonies characteristic of the subtilis group. The sirup
contained 96.84% sucrose and 1.21% invert sugar. The color
was 5, flavor 2, and score 850; depreciation from control, color
3, flavor 1, and score 125.

53. Number 53 was inoculated with gray yeast CLXXI.
A milky type of souring developed. The organism was recovered
in association with bacteria of the subtilis and fluorescent groups.
The plates showed a count of 630,000 organisms per cc. The
reaction was 6% N/100 acid. The sirup contained 92.13%

Micro-organisms oiJ Maple Sap 369

sucrose and 3.96^ invert sugar. The color was 6, flavor 3, and
score 750; depreciation from control, color 4, flavor _\ and

score 225.

54. Number 54 was inoculated with pink yeast ( WW I.
A deep milky appearance developed to a pronounced degree and
an unpleasant yeasty odor was noted. The organism was re-
covered, the plates showing- a count of 1,300,000 organisms per
cc. The reaction was 8.5% N/100 acid. The sirup contained
94.11% sucrose, and 2.71 % invert sugar. The color was 7,
flavor 22, and score 750; depreciation from control, color 5,
flavor iJ. and score 225.

55. Number 55 was inoculated with a non-fluorescent or-
ganism CVIII. A milky type of souring developed in which a
brownish color was evident in strong light, and a peculiar bac-
terial odor was noted. The organism was recovered in prac-
tically pure culture. The plates showed a total count of 7,230,-
000 organisms per cc. The reaction was 6% N/100 acid. The
sirup contained 94.76% sucrose and 2.38% invert sngar. The
color was 5, flavor 2, and score 850; depreciation from control.
color 3, flavor 1, and score 125.

56. Number 56 was the incubator control heated to boil-
ing and placed in the incubator with the other samples without
inoculation. It clouded slightly during the incubation period.
The plates poured revealed a bacterial count of 320,000 per cc.
The reaction was 3.1% N/100 acid. The sirup contained 96.029?
sucrose and 1.35% invert sugar. The color was 5, flavor 2, and
score 850; depreciation from control, color 3, flavor 1, and score

125-

57. Number 57 was the control not heated and retained

outside the incubator at low temperature during the incubation
period. At the time of evaporation it had a temperature of
io° C. The plates showed a development of 37,000 bacteria
per cc. The reaction was 2.5% N/100 acid. The sirup con-
tained 96.47% sucrose and o.6orr invert sugar. The color was
2, flavor 1, and score 975; depreciation from control (number

37(»

Bulletin 167

49), color o, flavor u, and score o. While this sample was given
the same score as number 49, it was considered by the expert
who scored the samples to be vcr\ slightly its inferior.

SERIES 7 : SIRUPS 58 TO 65

The sap for this series ran on March 21. It was collected
with the general supply in a gathering- tub late in the evening,
strained into buckets and allowed to remain over night in a cold
place. ( )n the morning of the following day all but one of the
samples was heated to boiling and cooled at once by placing the
buckets in ice water. As soon as cool the material was placed in
the incubator and cultures of organisms were added to the sample-
designed for inoculation. The air temperature maintained dur-
ing the incubation period appears on the accompanying graph
(figure 8).

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Fig. 8. Graph of incubation temperatures for series 7 ; saps 5S to 65 111-
clusive. The arrow heads on next to the bottom line from left to right indi-
cate respectively the time of inoculation, the time of evaporation of the first
sample and the' time of evaporation of the last sample.

58. Number 58 was a control evaporated on the morning
of .March 22. The plates developed 380 colonies per cc, among
which those of the fluorescent bacteria predominated. The reac-
tion was 3', X/100 acid. The sirup contained 97.01% sucro>c
and 0.43% invert sugar. The color was 3, flavor 1, and score 950.
This sample was regarded by the judge as the third best sirup
obtained during the entire series of experiments.

59. Number 59 was inoculated with red yeast CXXI1I. A
brownish-white clouding resulted. The organism was recovered
in association with spore-bearing bacteria apparently belonging
to the subtilis group. The plate< showed a development of
325.000 colonies per cc. The reaction was 8< c N/100 acid. The
sirup contained 94.74'' sucrose and 2.03$ invert sugar. The color

Micro-organisms oi- aI ai-i.i-: Sap 371

was 7, flavor 3, and score 725; depreciation Erom control, color ).
flavor 2, and score 225.

60. Number (>o was inoculated with red yeasl CXIII. A
milky brown souring developed. The organism was recovered in
association with bacteria belonging to the fluorescenl and subtilis
groups. The count showed 1,950,000 colonies per cc. The reac-
tion was 8.4% N/100 acid. The sirup contained 95.44% sucrose
and 1.76% invert sugar. The color was 6, flavor 3, and score
750; depreciation from control, color 3, flavor 2, and score 200.

61. Number 61 was inoculated with red yeast CX. A red-
dish-brown clouding developed. The organism was recovered,
the plates showing a count of 650,000 colonies per cc. among
which those of the introduced organism predominated. The re-
action was 5.5% N/100 acid. The sirup contained 95.80'.
sucrose and 1.77% invert sugar. The color was 9, flavor 3, and
score 675 ; depreciation from control, color 6, flavor 2, and
score 275.

62. Number 62 was inoculated with red yeast 25. A
reddish clouding developed accompanied by a sour yeasty odor.
The organism was recovered in association with bacteria,
050.000 colonies per cc. being developed of which practically one-
third were those of the introduced organism. The reaction
was 9.5/r N/100 acid. The sirup contained 89.27^ sucrose and
7.30% invert sugar. The color was 9, flavor 4, and score 550;
depreciation from control, color 6, flavor 3, and score 400.

63. Number 63 was inoculated with red yeast CXIV. The
sap became cloudy with a reddish-brown hue. Out of the
975,000 colonies per cc. which developed on the plates, 175,000
were characteristic of the introduced organism. The reaction
was 11% N/100 acid. The sirup contained 89.06^ sucrose and
7.O3CY invert sugar. The color was 8, flavor 3, and score 700;
depreciation from control, color 5, flavor 2, and score 250.

64. Number 64 was inoculated with red yeast CXX. A
sour yeasty odor developed and the sap became deeply clouded
with a reddish cast. The organism was recovered. The plates

372 Bulletin 167

showed a development of 97,500 colonies per cc., most of which
were characteristic of the introduced organism. The reaction
was 10% N/100 acid. The sirup contained 91.64% sucrose
and 5.86% invert sugar. The color was 8, flavor 3, and score
700; depreciation from control; color 5, flavor 2, and score 250.

65. Number 65 was a control retained in the incubator.
The sap clouded early and appeared like the inoculated samples.
The plates showed a development of 1,950,000 colonies of vari-
ous species. The reaction was 9% N/100 acid. The sirup
contained 91.89% sucrose and 3.52% invert sugar. The color
was 6, flavor 4, and score 625 ; depreciation from control (num-
ber 58), color 3, flavor 3, and score 325.

influence; of the container upon the quality of the sirup

Under the above heading will be discussed the sirups made
from the saps previously referred to in connection with the
discussion of the influence of the container upon the bacterial
content of the sap (page 343). It will be remembered that
six trees were employed which had been tapped with special
rare and upon which tin buckets were hung. For the purpose
of a part of this experiment, they were replaced by wooden
"ins. The tin buckets were kept free from the accumula-
tion of micro-organisms by frequent washings and scaldings.

66. Number 66 was made from sap collected -March 21
and 22, in clean tin buckets. The composite sample from the
6 trees contained 140 organisms per cc, most of them of a single
type previously mentioned as characteristic of tree 3 (Plate VI >.
The reaction was 2% N/100 acid. The sirup contained 96.62%
sucrose and 0.41^' invert sugar. The color was 3, flavor I, and
score 950.

67. Number 67 was made from sap obtained from the same
trees as was number 66 but it flowed two days later on March 23
and 24. The tin buckets were replaced by wooden buckets
formerly employed in the sugar place where the work was done.
They had been soaked out in the usual way and thoroughly

Micro-organisms of Maple Sap

washed before being" hung on the trees. That the crevices and
interstices of the wood afforded ample lodging places for micro-
organisms is readily seen from the fact that 6,500,000 colonies
per cc. developed from the mixture of sap obtained, and only a
few of them were characteristic of the organism from tree 3
(Plate VII). The reaction was 5.5% N/100 acid. The sirup
contained 88.25% sucrose and 6.21% invert sugar. The color
was 9, flavor 4, and score 550; depreciation from control (number
66), color 6, flavor 3, and score 400.

series 8: sirups 68 to 73

The sap of this series ran on March 22, and was collected
in the evening and stored over night in buckets, at a tempera-
ture slightly above its freezing point. The following morning the
samples were thoroughly mixed, and all but one were heated
to boiling and allowed to cool in water. At 3 p. m., they were
placed in the incubator and inoculated. The temperature main-
tained during the three days' incubation period may be seen by
reference to the graph (figure 9).

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Fig. 9. Graph of incubation temperatures for series 8 ; saps 68 to 73 in-
clusive. The arrow heads on next to the bottom line from left to right indi-
cate respectively the time of inoculation, the time of evaporation of the first
sample and the time of evaporation of the last sample.

68. Number 68 was a control boiled down on the morning
of March 23. Just before evaporation it contained 500 organ-
isms per cc. The reaction was 2.5% N/100 acid. The sirup
contained 96.90% sucrose and 0.51% invert sugar. The color
was 4, flavor I, and score 925.

69. Number 69 was inoculated with fluorescent organism
CLIV. It was recovered in practically pure culture from the
green sour sap which resulted. The plates showed a count of

374 Bulletin 167

19,500,000 organisms per cc. The reaction was 10% N/100
acid. The sirup contained 93.87% sucrose and 1.59% invert
sugar. The color was 9, flavor 3, and score 675 ; depreciation
from control, color 5, flavor 2, and score 250.

70. Number 70 was inoculated with green fluorescent or-
ganism CXXXIII. A greenish-brown souring developed prompt-
ly and the organism was recovered in practically pure culture.
The plates showed a count of 20,400,000 per cc. The reaction
was 10% N/100 acid. The sirup contained 93.47% sucrose and
2.46' , invert sugar. The color was 11, flavor 4, and score 500;
depreciation from control, color 7, flavor 3, and score 425.

71. Number 71 was inoculated with green fluorescent or-
ganism CXI All. A greenish-white clouding appeared within a
day. The plates showed a development of 23,400,000 organisms
per cc, practically all of which were of a type characteristic
of the introduced species. The reaction was 6.5% N/100 acid.
The sirup contained 93.69% sucrose and 0.58% invert sugar.
The color was 14, flavor 4, and score 425 ; depreciation from
control, color 10, flavor 3, and score 500.

72. Number 72 was inoculated with green fluorescent or-
ganism CLII. A green souring occurred. The plates showed a
development of 29,250,000 colonies per cc, practically all of
the fluorescent type. The reaction was 10% N/100 acid. The
sirup contained 95.07%) sucrose and 0.87' < invert sugar. The
color was 13, flavor 4, and score 450. Depreciation from con-
trol, color g, flavor 3, score 475.

/T,. Number 73 was inoculated with green fluorescent or-
ganism CLVIII. The sap promptly clouded, becoming at firsl
greenish and then milky. The plates showed a development of
J3.400.000 colonies per cc with pronounced green fluorescence.
The reaction was 2.$',', N/100 acid. The sirup contained 1)7.4;
sucrose, and o.(>N', invert sugar. The color was 6, flavor 2, and
seme 825; depreciation from control, color 2, flavor 1, and score

TOO.

M [CR0-0RGA1N [SMS OP M \ri i; S VP

si:k i i:s 9: sirups j.\ to 8]

The sap for this series was gathered in pails and broughl
to the laboratory about to p. m., March 24. It was carefully mixed
and divided into 18-quart portions, all but one of which were
heated to boiling. The following morning the samples which
had been heated were placed in the incubator and inoculated.
with the exception of one which was reserved as an incubator
control. The air temperature maintained during the incu-
bation period may be seen by referring to the accompanying
graph (figure eo).

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Fig. 10. Graph of incubation temperatures for series 9 ; saps 74 to si in
elusive. The arrow heads on next to the bottom line from lefl to ri^'lii indl
cate respectively the time of inoculation, the time of evaporation of the firsl
sample and the time of evaporation of the last sample.

74. Number 74 was the control which was not heated. It
was evaporated on the morning of March 25. The sap contained
750 organisms per cc. The reaction was 3% N/100 acid. Un-
fortunately the sirup was slightly burned in the pan so that its
color and flavor were seriously impaired. For this reason the
sirups of this series have been referred to number 68 as a con-
trol. Sirup 74 contained 96.36% sucrose and 0.70' J invert sugar.
The color was 9, flavor 4, and score 550; depreciation from con-
trol (number 68), color 5, flavor 3, and score 375.

75. Number 75 was inoculated with fluorescent organism
CLXXIX. The plates showed a development of 10.400,000
colonies per cc. divided between those characteristic of the
fluorescent group and those of another organism of the subtil is
type. The reaction was 10% N/100 acid. The sirup contained
84.74% sucrose and 10.77% invert sugar. The color was 9, flavor
3. and score 675 ; depreciation from control, color 5, flavor 2.
and score 250.

3'76 I'.ii.i.piTiN 167

76. Number 76 was inoculated with fluorescent organism
CLV. The organism was recovered, but in association with
great number of the spore-bearing bacteria noted in connection
with the previous sample. The plates showed a count of
4,875,000 organisms per cc. The reaction was 10.7% N/100 acid.
The sirup contained 87.03% sucrose and 8.94% invert sugar.
The color was 10, flavor 3, and score 650; depreciation from con-
trol, color 6, flavor 2, and score 275.

yy. Number yy was inoculated with green fluorescent or-
ganism CLXXVII. 11,700,000 colonies per cc. were obtained.
These were divided between the two types of organisms men-
tioned for the preceding samples of this series. The reaction
was [0.1% N/100 acid. The sirup contained 88.13% sucrose
and 8.24% invert sugar. The color was 8, flavor 3, and score
700; depreciation from control, color 4. flavor 2, and score
225.

78. Number 78 was inoculated with green fluorescent or-
ganism XXXVI. Here again two types of colonies were re-
covered. The total count was 14,625,000 colonies per cc. The
reaction was 1 1.2% N/100 acid. The sirup contained 83.86%
sucrose and 10.84'; invert sugar. The color was 8, flavor 4,
and score 575 ; depreciation from control, color, 4, flavor 3,
and score 350.

79. Number 79 was inoculated with non-fluorescent organ-
ism CLXIII which itself belongs to the subtilis group. 6,500,000
colonies per cc. were recovered, practically all of which resem-
bled those of the introduced organism. The reaction was 25.4' -
N/100 acid. The sirup contained 78.78% sucrose and 15.25
invert sugar. The color was 7, flavor 4, and score 600; deprecia-
tion from control, color 3, flavor 3, and score 325.

80. Number 80 was inoculated with pink yeast CLXVIII.
The organism was not recovered. 9,750,000 colonies were ob-
tained per cc, all of which were characteristic of the subtilis type.
The sirup contained 79.20% sucrose and 16.13% invert sugar.

Micro-organisms of M apu: Sap 377

The color was u>. flavor 4, and score 525; depreciation from
control, color 6, flavor 3, and score 400.

81. Number 81 was a control heated to boiling and left in
the incubator without inoculation. It developed 3,250,000 colo-
nies per cc, typical of the subtilis group. The reaction was m',
N/100 acid. The sirup contained 89.23% sucrose and 7.51s
invert sugar. The color was 7, flavor 4, and score 600 ; deprecia-
tion from control (number 68), color 3, flavor 3, and score 325.

It is evident that the sap used in the above series was
heavily contaminated by some spore-bearing organism which
apparently has the power of inverting sugar and which exercises
a further detrimental influence upon the flavor of sirup. Refer-
ence to the laboratory notes shows that after being heated the
samples were placed without being covered on the top of instead
of within the incubator to cool over night, in order that they
might be ready for inoculation the following morning; but
to prevent the entrance of falling dust a shelter of new clean
paper was supported a few inches above the tops of the buckets.
During the night there occurred a heavy wind storm. The
ground was partially bare and it is possible that spore laden dust
entered the laboratory through the rather large cracks which
had opened in the single layer of boards constituting the floor,
and that infection was produced in this way. Unfortunately the
infection was not suspected until the results of the bacteriological
and chemical analyses were known. Cultures of the organism
were not secured so that its identity is uncertain, but the agar
plate colonies were strikingly similar both in macroscopic and
microscopic appearance to those of Bacillus subtilis and spore
formation was apparent on unstained preparations from 4 days'
agar colonies.

SUGAR AND SIRUP FROM SOUR SAP

The inoculation work of the season was completed with the
preceding series, but a few experiments were made with natural

sour sap.

378 Bulletin 167

82. Number 82 was made from sap collected March 29
from two trees located in a sheltered position which received an
abundance of sunlight. The sap obtained from these trees
naturally soured somewhat earlier than the average sap in the
orchard as a whole and was just about ready to stop running- at
the time this experiment was started. The sap had been allowed to
remain without being collected since the first indications of serious
souring, 2 or 3 days before. Neither titration nor bacteriological
count was made. The sap was concentrated to sugar at once, to
see whether it would grain properly, which it did. The sample
was neither scored nor analyzed.

83. Number 83 was made from sap collected April 2 and
represented all the sap that had flowed from the trees mentioned
under 82, since March 29. At the time of collection these trees
had apparently entirely ceased running. The sap contained
43,875,000 organisms per cc. The reaction was 6% N/iooacid.
Like the preceding sample this was evaporated at once to a sugar
which grained readily. It was neither scored nor analyzed.

84. Number 84 was made April 4 from a part of the same
sap from which 83 was made. The sirup contained 91.14' «
sucrose and 2.83% invert sugar. The color was 20, flavor 3',
and score 350.

85. Number 85 was made from sour sap collected April 2,
but winch had been accumulating: in the buckets for several days.
It contained 11,275,000 organisms per cc. The reaction was 6' i
N/lOO acid. The sirup contained 94.68$ sucrose and T.57' !
invert sugar. The color was darker than 20, flavor 31, and score

325-

86. Number 86 had exactly the same history as 85 except
that when nearly evaporated to sirup it was allowed to cool to
about 350 C, following which the beaten white of two eggs
was added in order to test the clarifying power of this treatment.
The boiling was then continued. The sirup contained 93.21$
sucrose and 1.23% invert sugar. The color was darker than

Micro-organisms oj? Maple Sap ;'7'.»

jo but not quite as dark as that of number 85. The flavor was
31, and score 325.

SIRUP FROM LAST RUN SAP IN I9IO

The plan pursued in the following experiments was exactl)

the same as that described for similar experiments the previous
year (page 357). The first trees were retapped April 3. Il
was the intention to continue the experiments upon different
trees until the flow of sap was so reduced that it was impossible
to secure enough to make even a small sample of sirup. Unfor-
tunately for the purpose of the work the weather became warm
and night freezes ceased altogether, so that only a single experi-
ment of this character was possihle.

87. Number 87 was secured from the fresh tap-hole of a
tree which had yielded sour sap for a considerable number of
days and upon which the leaf buds were already opened. The
sap contained 5 organisms per cc. The reaction was 2.5'/ N/100
acid. The sirup contained 97.27% sucrose and 0.26% invert
sugar. The color was 4, flavor 5, and score 525; depreciation
from first run control, color o, flavor 3, score 250.

88. Number 88 was made from the same tree as number 87
but from the sap flowing from the old tap-hole. The plates gave
a count of 73,125,000 organisms per cc. The reaction was 4%
N/100 acid. The sirup contained 94.56% sucrose and r.79% in-
vert sugar. The color was 11, flavor 6, and score 250; deprecia-
tion from control (number 87), color 7, flavor 1, and score 275.
.Depreciation from first run control, color 7, flavor 4, score 625.

The results afforded by these two samples arc interesting
and significant, for it will be ohserved that the flavor of the last
inn material in 1910 was very seriously impaired, even when bac
teria were excluded, the typical buddy flavor being present in an
unmistakable degree, while the color remained light. Manifestly
the poor flavor of No. 87 can not be attributed to the influence
of the bacteria present for they were practically absent, but 5 per
cc. being found. Its cause must be sought elsewhere ; and naturally

380 Bulletin 167

one suspects physiological changes occurring- within the tree.
These results do not agree with those obtained in 1909, and cited
in bulletin 151, as well as on pages 357-358 in this issue; but
the contradiction may probably be explained by the divergen-
cies in the prevailing weather conditions during the two sea-
sons. The spring of 1910 was interrupted by periods of warm
weather which started the trees into vegetative activity at
least two or three weeks before the close of the sugar season,
whereas the season of 1909 was short and exhibited no warm
periods intervening with cold spells.

The small amount of invert sugar present in No. 87 con-
firms the suggestion obtained from the results of 1909, that the
invert sugar content of a sap tends to decrease rather than in-
crease as the season advances.

Inoculation Experiments in 191 i

The considerable number of instances in which the plates
from incubated samples failed to return approximately pure cul-
tures of the introduced organism in 1910, led to a modification
of the procedure for 1911. The incubation buckets were dis-
carded and a large number of 2 quart glass preserving jars were
secured in which the sap was placed and subjected to fractional
sterilization before inoculation. The covers were left on the
jars but were not clamped down, so that the protection from in-
fection was approximately the same as is afforded in Petri dish
cultures. For purposes of sterilization, the jars were placed in
a steam chamber and treated as is customary in sterilization with
flowing steam at atmospheric pressure. Except in the last series
of the season tbe sap was subjected to two sterilizations only.
The last series was steamed on each of three consecutive days.
This change was made because one of the jars of the control of
the preceding series developed cloudiness, which was found upon
examination to be due to spore-bearing organisms of the subtilis
type. As in the preceding season, the success of the inoculation
experiments was controlled by bacteriological examination in

Micro-organisms oi? Maple Sap

381

which the poured plate method was employed, but aliquots were
not used and no count of the number of recovered organisms
was obtained.

The saps were mixed and titrated immediately before con-
centration, plates were poured to determine the character of
the infection, and a sample was removed and sterilized for the
purpose of the experiment previously mentioned. The sirups
were handled in all respects like those of the preceding year.

series 10: sirups 89 to 96

The sap for this series flowed on March 22 and was gathered
and given the first sterilization late in the afternoon of that day.
The second sterilization followed after an interval of 24 hours.
The samples were inoculated as soon as they were sufficiently
cool, and were incubated for y/2 days. The air temperature
maintained in the incubator is shown in the accompanying graph
(figure 11).

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J

Fig. 11. Graph of incubation temperatures for series 10; saps 89 to 90 in-
clusive. The arrow heads on next to the bottom line from left to right indi-
cate respectively the time of inoculation, the time of evaporation of the first
sample and the time of evaporation of the last sample.

89. Number 89 was a control boiled down the day it was
obtained from the trees. The reaction was 1.2% N/100 acid. The
sirup contained 95.81% sucrose and 0.34^ invert sugar. The
color was 3, flavor 2, and score 900.

90. Number 90 was inoculated with Bacillus aceris, a
stringy sap organism, strain CCXVII. A very deep milky type
of souring occurred and the material became quite stringy,
though it was by no means as ropy as unsterilized sap inoculated
with the same organism. An unpleasant yeasty odor was pro-

382 Bulletin 167

nounced. The reaction was 25.4% N/100 acid. The sirup con-
tained 86.72% sucrose and 7.20% invert sugar. The color was
7, flavor 4, and score 600; depreciation from control, color 4,
flavor 2, and score 300.

91. Number 91 was inoculated with fluorescent organism
CXL. A characteristic green type of souring developed and the
organism was recovered. The reaction was 1.5% N/100 acid.
The sirup contained 94.76% sucrose and 0.40% invert sugar.
The color was 6, flavor 3, and score 750; depreciation from
control, color 3, flavor 1 and score 150.

92. Number 92 was inoculated with fluorescent organism
CXI I. A green type of souring developed and the organism
was recovered. The reaction was 1.4' < N/100 acid. The simp
contained 05.00', sucrose and 0.52$ invert sugar. The color
was 7, flavor 3, and score 725: depreciation from control, color
4, flavor 1, and score 175.

93. Number 93 was inoculated with green fluorescent or-
ganism LI. Typical green souring developed and the organism
was recovered. The reaction was 1.2' < X/100 acid. The sirup
contained 95.43$ sucrose and 0.43 invert sugar. The color was

6, flavor 2, and score 825; depreciation from control, color 3,
flavor o, and score 75.

94. Number 94 was inoculated with green fluorescent or-
ganism CXLI. Green souring developed and the organism was
recovered. The reaction was 1.2% N/100 acid. The sirup con-
tained 94.68%; sucrose and 0.52^ invert sugar. The color was

7, flavor 4, and score 600; depreciation from control, color 4.
flavor 2, and score 300.

95. Number 95 was inoculated with Bacillus aceris, a
stringy sap organism, strain LXXXY1I. A milky type of sour-
ing with slight stringiness developed. The organism was re-
covered. The reaction was 27% N/100 acid. The sirup con-
tained 90.85$ sucrose, and 5.04% invert sugar. The color was
6, flavor 3, and score 750; depreciation from control, color 3,
flavor I, and score 150.

Micro-organisms of AFait.k Sap

383

')<>. Number 96 was the control sterilized and placed in
the incubator without inoculation. The plates were sterile. The
reaction was 0.5% N/100 acid. The sirup contained 95-33^5
sucrose and 0.44', invert sugar. The color was 5, flavor 2, and
score 850; depreciation from control (number 89), color 2, flavor
o. and score 50.

series [i : sirups 97 to 104

The sap of this series was obtained on March 26, It was
sterilized at once and again on March 2j, and inoculated in the
evening- of the latter day and incubated for 3^ days. The
temperature is shown in the accompanying graph (fig. 12).

c

JL

J-l

, ,

l_i

u

1

u

_J

-

' i

r

. ■

L-.

1

1

,

>

■

•

■

■

1

j

U-fl

j^

t

\

I

\

'

1

fl

\

■1

1

V

1,

-]

/

u

es

V

->

J

/

i

!

7

Fig. 12. Graph of incubation temperatures for series 11 ; saps 97 to 104 in-
clusive. The arrow heads on next to the bottom line from left to right indi
eate respectively the time of inoculation, the time of evaporation of the first
sample and the time of evaporation of the last sample.

97. Number 97 was a control evaporated March 26 im-
mediately after gathering. The reaction was 0.5% N/100 acid.
The sirup contained 96.04% sucrose and 0.19% invert sugar.
The color was 3, flavor 1, and score 950.

98. Number 98 was inoculated with pink yeast CXXXII.
A milky clouding developed accompanied by a very unpleasant
odor. The organism was recovered. The reaction was 1.5',
N/100 acid. The sirup contained 86.70^ sucrose and S.Oo' -
invert sugar. The color was 9, flavor 4. and score 550; de-
preciation from control, color 6, flavor 3, and score 400.

99. Number 99 was inoculated with red yeast CX. A red-
dish brown type of souring developed, accompanied by a yeast)
odor, 'ldie organism was recovered. The reaction was 1.4',
N/100 acid. The simp contained 94.82^? sucrose and 2.05',

384 Bulletin 167

invert sugar. The color was 7, flavor 4, and score 600 ; depreci-
ation from control, color 4, flavor 3, and score 350.

100. Number 100 was inoculated with fluorescent organ-
ism CXLVIII. A green type of souring developed and the or-
ganism was recovered. The reaction was 1.1% N/100 acid.
The sirup contained 96.21% sucrose and 0.93% invert sugar.
The color was 7, flavor 3, and score 725 ; depreciation from con-
trol, color 4, flavor 2, and score 225.

101. Number 101 was inoculated with fluorescent organism
5. A characteristic type of souring appeared and the organism
was recovered. The reaction was 1.2% N/100 acid. The sirup
contained 96.45% sucrose and 0.94% invert sugar. The color was
8, flavor 3, and score 700; depreciation from control, color 5.
flavor 2, and score 250.

102. Number 102 was inoculated with green fluorescent
organism XXXIII. A green type of souring developed and the
organism was recovered. The reaction was 4.5'' X /Too acid.
The sirup contained 95. 6j(A sucrose and 1.14'' invert sugar.
The color was 7, flavor 4, and score 600; depreciation from con-
trol, color 4, flavor 3, and score 350.

103. Number 103 was inoculated with green fluorescent or-
ganism XXXVI. A green type of souring developed and the
organism was recovered. The reaction was i.4rr N/100 acid.
The sirup contained 95. 38',' sucrose and 1.35'' invert sugar.
The color was 7, flavor 4, and score 600; depreciation from con-
trol, color 4, flavor 3, and score 350.

104. Number 104 was a control sterilized and placed in
the incubator without inoculation. The plates were sterile. The
reaction was 0.6% N/100 acid. The sirup contained 97.0
sucrose and 0.41'- invert sugar. The color was 4. flavor 2. and
score $J$\ depreciation from control (number 07), color t, flavor
1, and score 75.

-

I
a;

£

i

Plate IV. — Incubator, i See pages 359-360).

Micro-organisms of Maitm Sap

385

SERIES 12 : SIRUPS [05 l" I [2

The sap for this series was collected on March 27, given
two sterilizations, inoculated on the morning of March 29, and
incubated for 3 days at the temperatures indicated in the accom-
panying graph (figure 13).

O 3 f 3 'i. IS '« M 3f J7 XI

42 1S <a Si 14 37 SO ii ££ ?3 72 7} y 61 e-r 87 X 91 3f 3t i&z '15 iff 'ii i'* '» ^ h.

s

h

\

U'

Yk

r

Fig. 13. Graph of incubation temperatures tor series 12 : saps 105 to 1 111
inclusive. The arrow heads on next to the bottom line from left to right iinli
cate respectively the time of inoculation, the time of evaporation of the first
sample and the time of evaporation of the last sample.

105. Number 105 was a control evaporated on the morning
of March 28. The reaction was 1% N/100 acid. The sirup
contained 96.23% sucrose and 0.24 invert sugar. The color was
3, flavor 2, and score 900.

106. Number 106 was inoculated with red yeast CCIII,
which evidently made a feeble growth as the organism was not
recovered from the rather high dilutions employed in plating.
The reaction was 4.1% N/100 acid. The sirup contained
97.30% sucrose and o.66c'c invert sugar. The color was 7, flavor
3, and score 725 ; depreciation from control, color 4, flavor 1, and
score 175.

107. Number 107 was inoculated with pink coccus CYI.
A pale milky type of souring developed. The organism was
recovered. The reaction was 4% N/100 acid. The sirup con-
tained 94.52% sucrose and 0.69% invert sugar. The color was
5, flavor 4, and score 650; depreciation from control, color 2,
flavor 2, and score 250.

108. Number 108 was inoculated with pink yeasl
CLXXVIII, but only one colony was recovered. The reaction
was 3.3% N/100 acid. The sirup contained 95.37% sucrose and

386 Bulletin 167

0.90'/ invert sugar. The color was 4. flavor 3. and score 800:
depreciation from control, color I, flavor 1. and score 100.

109. Number 109 was inoculated with gray yeast 24. The
organism made a remarkahle growth as evidenced by the deep
clouding of the material and a flaky sediment, as well as by the
semi-mycelial growth suspended in the sap. A distinctly yeasty
odor developed which hecame very offensive when the material
was heated for evaporation. The specific organism was recovered
in great numbers. 'The reaction was 41 X XX'ioo acid. The
sirup contained ^J.j^f sucrose and 2^.2^', invert sugar. The
color was 10, flavor 4, and score ^2^: depreciation from control.
color 7, flavor 2. and score i,y^.

no. Number no was inoculated with gray yeast CXXI.
The organism was recovered from the material which developed
a dull brownish white appearance with clouding. The reaction
was 3.3^ X 100 acid. The sirup contained 07.0(1', sucn
and 1.74X invert sugar. The color was 6, flavor 4. and score
625; depreciation from control, color 3, flavor 2, and score 275.

1 it. Number 111 was inoculated with green mold CCCI
belonging to the Eurotium genus. The organism made moder-
ate growth as could he seen by the tufts of mycelium developing
in the material and also from the plates. The reaction was 2' ,
X 100 acid. The sirup contained 1)3.31 X sucrose and 4.30
invert sugar. The color was 7. flavor 4. and score 600: depreci-
ation from control, flavor 4. color 2. and score 300.

112. Number 112 was the control sterilized and held in the
incubator without inoculation. The reaction was 1% X mo
acid. The sirup contained 97.60^ sucrose and 1.11X invert
sugar. The color was 5, flavor 2, and score 850; depreciation
from control (number 105), color 2, flavor o. and score 50.

SERIES 13 : sirups 1 13 to 120

'I "he sap for this series (lowed on March 30. and was stored
outdoors over night at a temperature below freezing, as shown
by (lie ice which formed in the cans during: the night. The

MlCRO-OKGANlSMS OF MA1XE Sac

material was sterilized on the mornings of .March 31, April 1 and
April 2, inoculated when cool, and incubated at temperatures in-
dicated in the graph (figure 141.

1_.

«

_ii-

— 1

1 ,

A

J -

n

l 1

I i

■>.

« )

1

..

* ■

^

1

■ 1

:

. 1

.

J-i

7 ] ;

iiuiii

^~ <

— ,

_'_*_

..

—

f

\

A

1

N

\

V

-sj

s

\

\

/"

s,

1

-/

I

\

,'

\

*\

V

1

I

^ v

/}

/

\

\

J

'

A

/

\

/

J

r-\

*-»

-'

a

1 '7 -\< : \ f t /■ 4 r 1 t'A .

5.1-

1

>M

**

;.'

/

^

(il

-^

~>

,-'

^

i

N

^

?«

^

\

/J

Fig. 14. Graph of incubation temperatures for series 13 : saps 11:: in L20
inclusive. The arrow heads mi next 1" the bottom line from left \<> righl indi
cate respectively the time o£ inoculation, the time <>f evaporation of the Brs1
sample ami the' time of evaporation of the last sample.

113. Number 113 was a control evaporated on the morning
of March 31. The reaction was 2% X/100 acid. The sirup
contained 96.97% sucrose and 0.61'. invert sugar. The color
was 4. flavor 2, and score 875.

114. Number 114 was inoculated with red yeast LXII.
A reddish type of clouding developed and the organism was re-
covered in large numbers. The reaction was 5/, X 100 acid.
The sirup contained 96.31 ''/< sucrose and i.oiA invert sugar.
The color was 5, flavor 3, and score J~$\ depreciation from con-
trol, color 1, flavor 1, and score 100.

115. Number 115 was inoculated with gray yeast CXXI.
A milky type of souring developed. The organism was re-
covered. The reaction was 4.7'. X 100 acid. The sirup con-
tained 95.14X sucrose and 1.52X invert sugar. The color was
7, flavor 4, and score 600; depreciation from control, color 3,
flavor 2, and score 275.

116. Number 116 was a composite inoculated with red
yeast LXII and fluorescent pseudomonas CXLV. A green type
of souring developed. The fluorescent organisms appeared in
large numbers on the plates, hut the yeast was not recovered.

388 Bulletin 167

The reaction was 3.7% N/100 acid. The sirup contained 95.62
sucrose and 0.68% invert sugar. The color was 8, flavor 4, and
score 575 ; depreciation from control, color 4, flavor 2, and
score 300.

117. Number 117 was inoculated with green mold CCCI
belonging to the Eurotium genus. An excellent growth occurred.
Some of the tufts of mycelium rose to the top and developed
characteristic green spores. The reaction was 7.4% N/100 acid.
The sirup contained 91.09% sucrose and 3.72% invert sugar.
The color was 9, flavor 4, and score 550; depreciation from
control, color 5, flavor 2, and score 325.

118. Number 118 was a composite inoculated with gray
yeast CXXI and fluorescent pseudomonas CXLV. The bacteria
were recovered in great numbers in association with a few
yeast colonies. The type of souring was characteristic of the flu-
orescent organism, but there was also evidence of the odor which
is associated with the development of yeast. The reaction was
4.8% N/100 acid. The sirup contained 95.69% sucrose and
0.98% invert sugar. The color was 7, flavor 4, and score 600 ;
depreciation from, control, color 3, flavor 2, and score 275.

1 19. Number 1 19 was a composite inoculated with Eurotium
CCCI and fluorescent pseudomonas CXLV. The mold ap-
parently made only very slight growth, while the bacteria de-
veloped luxuriantly. The reaction was 4.2% N/100 acid. The
sirup contained 96.27% sucrose and 0.81% invert sugar. The
color was 7, flavor 2, and score 800; depreciation from control,
color 3, flavor o, and score 75.

120. Number 120 was an incubator control, sterilized and
held without inoculation. The reaction was 4.6%) N/100 acid.
The sirup contained 95.02% sucrose and 0.57% invert sugar.
The color was 5, flavor 3, and score 775; depreciation from
control (number 113), color 1, flavor 1, and score 100.

Micro-organisms oe Maple Sap 389

sirups from last run sap in i9ii
As in two preceding years, as soon as the season was suf-
ficiently advanced, trees which were running a sour sap were
selected to be retapped in order to procure material from both
fresh and sour tap-holes of the same tree at the same time.

121. Number 121 was made from sour sap obtained on
April 21, after the buds were beginning to show considerable
green. The sap was cloudy but not excessively so. The sirup
contained 93.94% sucrose and 0.34% invert sugar. The color
was 7, flavor 5, and score 450; depreciation from control (num-
ber 122), color 2, flavor o, and score 50; depreciation from the
first run control of the season (number 89), color 4. flavor 3.
score 450.

122. Number 122 was made from sap obtained from the
same tree as 121 and at the same time but from the fresh tap-
hole. The sirup contained 95.80% sucrose and 0.07', invert
sugar. The color was 5. flavor 5, and score 500; depreciation
from first run control, color 2, flavor 3, and score 400.

123. Number 123 was made from sap obtained April 24
from a sour tap-hole. The sirup contained 95.33$ sucrose and
0.64% invert sugar. The color was n, flavor 6, and score
250; depreciation from control (number 124), color 6, flavor 1.
and score 250; depreciation from first run control, color 8,
flavor 4, and score 650.

124. Number 124 was made from sap obtained from the
same tree and at the same time as that for 123, but from a fresh
tap-hole. It contained 98.54% sucrose and 0.59% invert sugar.
The color was 5, flavor 5, and score 500; depreciation from first
run control, color 2, flavor 3, and score 400.

125. Number 125 was made from sour sap obtained from
the tree April 24. It contained 93.88% sucrose and 0.53% in-
vert sugar. The color was 7, flavor 5, and score 450; depreci-
ation from control (number 126), color 1, flavor o. and score 25;
depreciation from first run control, color 4, flavor 3, and score

45°-

390 Bulletin i6y

126. Number 126 was obtained from the same tree ami
at the same time as number 125. but from a fresh tap-hole. The
sirup contained 94.46% sucrose and 0.31% invert sugar. The
color was 6, flavor 5. and score 475 : depreciation from first
run control, color 3, flavor 3, and score 425.

127. Number 127 was made from sour sap obtained April

27. The sirup contained 87.41 '< sucrose and 4-59r^ invert
sugar. The color was 15, flavor 6. and score 150: depreciation
from control (number 128), color 11. flavor I, and score 375:
depreciation from first run control, color 12, flavor 4, and score

750-

128. Number 128 was made from sap obtained from the

same tree at the same time a- that for 127 but from a fresh
tap-hole. The sirup contained 94.21' , sucrose and 0.31$ invert
sugar. The color was 4. flavor 5. and score 525: depreciation
from first run control, color 1. flavor 3, and score 375.

129. Number 129 was obtained from a fresh tap-hole April

28. It contained 95.34' < sucrose and 0.12', invert sugar. The
color was 5. flavor 5. and -core 500: depreciation from first
run control, color 2, flavor 3. and score 400.

130. Number 130 was made from sap obtained from the
same tree and at the same time as that for 129, but from the
sour tap-hole. It contained 93.80', sucrose and 1.34'' invert
sugar. The color was 12, flavor 6, and score 225: depreciation
from control (number 129), color 7, flavor 1. and score 2jy.
depreciation from first run control, color g, flavor 4. and score

The results of this series of experiments confirm those
obtained the previous year, indicating that, accompanying the
resumption of vegetative activity in the tree, there is a change
in sap which makes it impossible thereafter to produce sirups
of superior flavor.

Micro-organisms of M ai'i.k Sap 391

Statistical Summary of Field Experiments

For the sake of convenient reference a summary of the statis-
tical data presented in the preceding pages is appended in tabular
form and in numerical order (table 9). The number of the
sample, the character of the organism employed or of the sample,
tlie designation of the organism, the bacterial count per cc. of
the sap, and the reaction of the sap in percent of N/100 aeid
occupy the first five columns respectively. These are followed
by the percentages of sucrose and invert sugar in the sirups,
calculated to a dry matter basis. The numbers in the column
headed "ratio" were obtained by dividing the percent of sucrose
by the percent of invert sugar. A small ratio number, there-
fore, indicates a relatively high proportion of invert sugar and
a high number, a small proportion. The ratio numbers with their
corresponding invert sugar values are shown below in tabular
form :

Invert sugar values
above 15 ' -

15 ', 10 s>..',

M..', - -I'..',
4V, - 2 ',

2 >;< "i ',

1 ', - >,.',

¥>% " v<

below 1 1 ' ,

Ratio numbers

1 to

5

5 "

10

10 "

20

20 "

50

50 "

90

90 "

180

ISO "

400

above

400

Following the ratio column are given the values for color,
flavor and score, assigned to the various sirups, and also the de-
preciation in color, flavor and score of inoculated sirups from
the corresponding value assigned to their respective controls. A
date column completes the table.

392 Bulletin 167

TABLE 9. TABULAR SUMMARY OF STATISTICAL DATA

No.

Character of sample

Organism

or organism

number

Count

Reaction

Sucrose

1

Control,

96.84

2

Fluorescent,

XXXIII

96.79

O

Ditto,

XXXVI

96.73

4

Ditto,

L

96.54

5

Ditto,

LIII

97.16

G

Control,

95.41

7

Fluorescent,

LVI

96.16

S

Ditto,

5

97.14

9

Non-fluorescent,

XXVI

95.61

10

Ditto,

XXX

95.86

11

B. aceris,

LXXXVII

170.0

96.12

12

Stringy peas,

I

95.71

13

Gray yeast,

24

93.34

14

Red yeast,

25

9.0

95.30

15

Ditto,

LXII

5.0

93.26

1G

Penicillium,

LXIV

8.2

95.73

17

Ditto,

LXV

8.0

92.76

18

Red yeast,

LXXIX

12.0

91.65

19

Composite,

5.0

93.2S

20

Control,

2.0

95.37

21

Last run, sweet,

95.19

22

Ditto, sour.

95.27

23

Ditto, sour

92.56

24

Ditto, sweet,

96.02

25

Ditto, sour,

94.05

26

Ditto, sweet,

96.42

27

Control,

10000

96.19

28

Gray yeast,

24

437000

82. S3

29

Fluorescent,

CXXXIX

12350000

94.91

30

Ditto,

CLIX

32G2000

93.75

*31

Gray yeast,

CXXI

5667000

96.62

*32

Ditto,

CLXXX

5600000

95.91

*33

Ditto,

CLXVIII

1950000

95.61

34

Control, 40°

7312000

95.S6

35

Red yeast,

CXL

2255000

95.13

*36

Ditto,

CII

7312000

96.16

*37

Ditto,

CLXXV

8825000

95.88

38

Control, 80°

162500

94.S0

*39

Gray yeast,

C

9750000

96.18

40

Incubator control.

5850000

95.05

41

Control,

66

1.2

97.S2

42

Red yeast,

CVII

13650000

19.0

93.27

43

Control, 100°

1625000

6.5

94.19

44

Fluorescent,

CXLVIII

9000000

10.0

93.03

45

Incubator control,

195i mi

4.5

96.28

4G

Pink coccus,

CIV

14300000

10.0

95.07

"•'Infection failed.

Micro-organisms oi? Maple Sap 393

TABLE '.'. TABULAE MMMAKY 01 STATISTICAL DATA

Ratio

Color

Flavor

Score

DEPRECIATION

lnv. Sug.

Color

Flavor

Score

Date

1.07

90

7

1

850

1

0

25

3-24-09

1.55

62

14

4

425

8

3

450

it

2.09

46

11

2

700

5

1

175

i(

1.56

62

9

2

750

3

1

125

II

1.44

67

11

2

700

5

1

175

ft

0.97

98

6

1

S75

It

1.64

59

10

2

725

4

1

150

3-27-n:t

1.15

84

10

2

725

4

1

150

If

0.78

122

5

2

850

-1

1

25

ll

0.77

124

6

9

o

750

0

2

12.",

it

1.72

56

7

4

600

1

•>
o

275

it

2.23

43

6

o
o

750

0

2

12^

it

2.53

37

7

2

soo

1

1

75

4- 1-09

3.01

32

10

2

725

4

1

L50

(1

2.98

31

8

2

775

2

1

100

f l

2.61

37

10

4

525

4

O

350

it

3.47

27

10

2

725

4

1

150

II

3.12

29

10

2

725

4

1

150

a

4.09

23

9

3

675

o

2

200

a

1.58

60

6

1

875

it

0.82

116

3

1

950

4-12-09

2.76

35

8

3

700

5

2

250

II

3.55

26

14

3

550

7

2

300

If

0.61

157

7

1

850

If

3.13

30

14

3

550

9

2

350

4-16-09

0.51

189

5

1

900

fi

0.30

321

4

2

875

3- 7-10

12.15

7

11

4

500

7

2

375

ii

0.38

250

8

21

750

4

01

125

f f

0.89

105

8

2

775

4

0

100

ft

0.30

322

6

2

825

2

0

50

ft

0.46

208

6

2

825

2

0

50

ll

0.23

416

7

2

800

o

0

75

ll

0.42

228

6

21

800

2

01

75

ll

1.15

83

12

21

650

8

01

225

ll

0.61

158

9

21

725

5

01

150

ll

0.64

150

11

21

675

7

01

200

ft

0.48

198

5

21

825

1

0"

50

ft

0.26

370

7

2

800

9

f >

0

75

ft

0.64

149

9

2

750

5

0

125

If

0.45

217

4

1

925

3-1 HO

2.14

44

5

22

800

1

1"

L25

a

2.64

36

7

2

800

O

1

125

if

1.40

67

9

3

675

5

2

250

if

0.51

189

4

1

925

0

0

0

ft

1.05

91

6

2

825

2

1

100

ii

394

I'.ri.u'.TiN 167
table 9 — Continued

Jo.

Character of sample

Organism

or organism

number

Count

Reaction

Sucrose

47

Pink coccus,

IV

1950000

9.6

88.81

4S

Gray yeast,

CLXXX

1625000

16.7

93.89

49

Control,

100

1.0

96.51

50

Pink coccus,

XLIX

1950000

8.0

93.83

51

Ditto,

CVI

4225000

3.5

97.30

52

Gray yeast,

CLXII

1275000

6.0

96.84

53

Ditto,

CLXXI

630000

6.0

92.13

54

Pink coccus,

CXXVI

1300000

8.5

94.11

55

Non-fluorescent,

CVI 1 1

27::oooo

6.0

94. 76

56

Control, 100

320000

3.1

96.(12

57

Incubator control.

370IMI

2.5

96. 17

58

Control,

380

3.0

97.01

59

Red yeast.

CXXIII

:; 25000

8.0

94.74

60

Ditto,

CXI 1 1

L950000

s.4

95.44

61

Ditto,

ex

650000

5.5

95.80

62

Ditto,

25

650000

9.5

89.27

63

Ditto,

CXIV

975000

11.0

89.06

04

Ditto,

exx

97500

10.0

91.64

65

Incubator control,

1950000

9.0

91.89

66

Tin buckets,

140

2.0

96.62

67

Wood buckets,

6500000

5.5

SS.25

68

Control

500

2.5

96.90

69

Fluorescent,

CLIV

19500000

LO.O

93.87

70

Ditto,

CXXXIII

20400000

10.0

93.47

71

Ditto.

CXLVII

L':',4oonoii

6.5

93.69

72

Ditto,

CLIII

29250000

10.0

95.07

73

Ditto,

CLVIII

2:1400000

2.3

97.47

*74

Control burned.

750

3.0

96.36

*75

Fluorescent,

CLXXIX

10400000

10.0

84.74

*76

Ditto,

CLV

4875000

10.7

87.03

*77

Ditto,

CLXXVII

11700000

10.1

88.13

*78

Ditto,

XXX VI

14625000

11.2

83.86

*79

Non-fluorescent,

CLXIII

6500000

25,1

78.78

*80

Pink yeast,

CLXXVIII

975 in

12.5

79.2(1

*S1

Incubator control,

3250000

10.0

89.23

t82

Sour sap,

f83

Ditto,

1:1875000

6.0

84

Sour sap kept.

91.1 1

85

Ditto,

L1275000

6.0

94.68

86

Ditto,

11275000

6.0

93.21

87

Last run, fresh,

5

2.5

97.27

88

Ditto, sour.

73125000

4.0

94.56

89

Control,

1.2

95.81

90

B. aceris,

CCXVII

25.4

86 72

91

Fluorescent.

CXL

1.5

94.7(1

*A spore bearing organism with inverting power was present in
association with the introduced species.
fS2 and 83, made into sugar.

\l [CR( h iRG w [SMS <H' M \ri.K S \r

.".'.i;

TABLE 9 — Colli i )i in',1

Ratio

Color

Flavor

Score

DEPRE< l.\ 1 [ON

Inv. Sug.

Color

Flavin

Score

Date

7.90

11

6

•»

750

2

2

175

:; 1 1-10

2.35

Hi

6

22

775

2

1

150

. 4

0.42

230

2

1

975

3-20-10

2.20

43

6

21

800

4

r

175

"

0.65

150

4

2

875

2

l

100

•■

1.21

80

r

0

2

850

0

l

125

••

3.96

23

6

*>
■J

Ton

4

2

225

> (

2.71

35

7

2J

750

5

l2

225

• .

2.38

40

5

2

S5i»

0

1

125

L35

71

5

2

850

•1»

1

125

••

ii.iiii

161

2

1

975

0

i)

n

■■

0.43

226

1

950

3-21-10

2.03

47

7

*>
O

725

4

2

225

■■

1.76

54

6

•>
•i

750

3

2

200

<<

1.77

54

9

q

675

6

2

275

. .

7.30

12

9

4

550

6

3

1(10

<i

7.63

12

8

n

700

5

2

250

"

5.86

16

8

■ >
• ■

700

5

2

250

"

3.52

26

G

4

625

3

O

325

••

(1.41

236

1

950

3-22-10

6.91

13

9

4

550

6

■>

too

3-24-10

0.51

190

4

1

925

3-22-10

1.59

59

9

675

5

2

250

. >

2.46

38

11

4

500

7

•>
• )

425

• .

ll.oS

162

14

4

425

10

•>

500

it

0.87

109

13

4

450

9

•>
->

475

it

0.68

143

6

2

SI' 5

2

1

100

a

0.70

138

9

4

550

5

•>

o

3 75

3-24-1 n

10.77

8

9

675

5

2

250

8.94

10

in

•>

650

6

2

275

. .

8.24

11

8

3

700

4

2

225

((

10.4S

8

8

4

575

4

350

It

15.25

5

7

4

600

O
Q

3

3 25

«(

16.13

5

10

4

525

G

q

400

u

7.51

12

7

4

600

3

3

325

3-29-10
4- 2-ln

2.83

32

2ii

350

••

1.57

60

20*

•>1
O

325

i.

1.23

76

20*

*->

325

(<

0.26

374

4

5

525

4- 3,-1 n

1.7!)

53

11

6

250

—

1

275

••

0.34

282

O

•>

2

900

3-22-11

7.20

12

7

4

600

4

2

300

••

0.40

237

6

o

■ I

750

1

L50

*A spore bearing organism with inverting power was preseni in
association with the introduced species.

396

Bulletin 167
table 9 — Concluded

No.

Character of sample

Organism

or organism

number

92

Fluorescent,

CXII

93

Ditto,

LI

94

Ditto,

CXLI

95

B. aceris,

LXXXVII

96

Incubator control,

97

Control,

98

Pink yeast,

CXXXII

99

Red yeast,

CX

100

Fluorescent,

CXLVIII

101

Ditto,

5

102

Ditto,

XXXIII

103

Ditto,

XXXVI

104

Incubator control,

105

Control,

$106

Red yeast,

can

107

Pink coccus,

CVI

$108

Pink yeast,

CLXXVIII

109

Gray yeast,

24

110

Ditto,

CXXI

111

Eurotium,

CCCI

112

Incubator control.

113

Control,

114

Red yeast,

LXII

115

Gray yeast,

CXXI

116

Composite,

I LXII
t CXLV

117

Eurotium,

CCCI

118

Composite,

f CXXI
t CXLV

119

Composite,

J CCCI
I CXLV

120

Incubator control,

121

Last run sour

122

Ditto, sweet

123

Ditto, sour

124

Ditto, sweet

125

Ditto, sour

126

Ditto, sweet

127

Ditto, sour

128

Ditto, sweet

129

Ditto, sweet

130

Ditto, sour

Count

Reaction

Sucrose

1.4

95.00

1.2

95.43

1.2

94.68

27.0

90.85

0.5

95.33

0.5

96.04

1.5

86.70

1.4

94.82

1.1

96.21

1.2

96.45

4.5

95.67

1.4

95.38

0.6

97.00

1.0

96.23

4.1

97.30

4.0

94.52

3.3

95.37

41.0

67.33

3.3

97.09

2.0

93.31

1.0

97.60

2.0

96.97

5.0

96.31

4.7

95.14

3.7

95.62

7.4

91.09

4.8

95.69

4.2

96.27

6.4

95.02

93.94

95.80

95.33

98.54

93.88

94.46

87.41

94.21

95.34

93. S6

$106 and 108, infection failed.

Micro-organisms of M \ n.i; Sap 39'

table 9 — ConeliHh >1

Ratio

Color

Flavor

Score

DEPRECIATION

In v. Sug.

Color

Flavor

Score

Date

0.52

183

7

3

725

4

l

it:,

3-22-11

0.43

222

6

2

825

3

0

75

tt

0.52

IV'

7

4

600

4

2

300

(1

5.04

IS

6

O

0

750

o

1

150

II

0.44

217

5

2

s;,,i

2

0

50

((

0.19

505

3

1

950

3-26-1 1

8.G9

10

9

4

:,;,ii

6

3

400

tt

2.05

46

7

4

600

4

3

350

tt

0.93

103

7

3

725

4

2

225

"

0.94

103

8

3

700

5

2

250

ft

1.14

84

7

4

600

4

3

350

tt

1.35

71

7

4

600

4

3

350

a

0.41

237

4

2

875

1

1

75

it

0.24

403

6

2

900

3-27-11

0.6G

147

7

3

725

4

1

L75

"

0.G9

137

5

4

650

2

2

250

tt

0.90

106

4

3

800

1

1

100

tt

28.35

2

10

4

525

7

2

375

<<

1.74

56

6

4

625

o
O

2

275

it

4.39

21

7

4

600

4

2

300

tt

1.11

88

5

2

850

2

0

50

tt

0.61

159

4

2

875

3-30-11

1.01

95

5

3

775

1

1

100

a

1.52

63

7

4

600

3

2

275

tt

0.68

141

8

4

575

4

2

300

tt

3.72

25

9

4

550

5

2

325

a

0.98 98 7 4 600 3 2 275

0.81

119

7

2

800

O

0

75

tt

0.57

167

5

3

775

1

1

100

" *

0.34

276

7

5

450

2

0

50

4-21-11

0.67

143

5

5

500

tt

0.64

149

11

6

250

6

1

250

4-24-11

0.59

167

5

5

500

it

0.53

125

7

5

450

1

0

25

tt

0.31

305

6

5

475

tt

4.59

19

15

6

150

11

1

375

4-27-11

0.31

304

4

5

525

II

0.12

794

5

5

500

4-28-11

1.34

70

12

6

225

7

1

275

n

398 Bulletin 167

Discussion of Related Sirups in Groups

A Mirvey of the preceding- table shows that the sirups natu-
rally fall into different groups according to the character of the
sap used or of the treatment accorded to it. For convenient
study and discussion the tables have been re-arranged so as to
bring similar samples together in groups.

It appeared in the discussion of individual samples that the
inoculation failed in certain instances. Such samples have been
placed by themselves and are grouped together without regard to
the character of the organisms with which inoculation was at-
tempted. In one series a spore-bearing organism became at least
equally as important as the bacteria which were artificially intro-
duced, and this series has been treated as a unit in the re-arrange-
ment. The samples inoculated with yeasts and molds showed a
tendency to mixed infection in which the fluorescent organism^
naturally present played a more or less important part. With the
other statistical data in the following tables, the average color,
flavor and score for each group, as well as the average deprecia-
tion of color, flavor and score, is recorded at the foot in the
proper columns.

Twenty-two sirups were made from sap successfully inocu-
lated with one or another strain of fluorescent bacteria. The
average color was 9, flavor 2.0. and score 605 ; the average depre-
ciation from control, color 4.8, flavor 1.6, and score 242.

Four samples were successfully inoculated with non-fluo-
rescent bacteria. The average color was 5.5, flavor 2.5, and,
score 800: the average depreciation from control, color 0.5, flavor
JA. and score 100.

Seven samples were influenced by the action of a spore-
bearing organism of the subtilis type which appeared sponta-
neously. Four of these were inoculated with fluorescent organ-
isms, one with a non-fluorescent bacillus similar to the intruder,
and one with a pink yeast which was not recovered, while the
other was intended for an incubator control. It is evident that
the quality of these sirups must lie attributed to the combined
action of the two groups of organisms. The average color was

Micro-organisms of Maple Sap 390

8.4, flavor 3.6, and score 618; the average depreciation from

control, color 4.4, flavor 2.6, and score 307.

Three sirups were made from sap successfully inoculated
with Bacillus aceris, a stringy sap organism, of which two strains
were employed. The average color was 6.7, flavor 3.7 and score

650; the average depreciation from control, color 2.7, flavor _\
and score 242.

Six samples were inoculated with pink cocci belonging t« > the
Micrococcus roscus type. The average color was 5.7. flavor 2.6,
and score 775; the average depreciation from control, color 2.8,
flavor 1 .f>, and score 171 .

( )nly one sample was successfully inoculated with pink yeast.

Thirteen samples were inoculated with red yeast with at least
partial success. The fluorescent bacteria developed in associa-
tion with the introduced organisms in a considerable number of
these samples, so that the quality of sirup secured must he
assigned to the combined activities of the two species. The
average color was 8, flavor 2.8, and score 703; the average de-
preciation from control, color 4.1, flavor 1.7, and score 215.

Eight samples were inoculated with gray yeast with partial
or complete success. Here again the fluorescent bacteria some-
times developed in association with the yeasts and the results
must be regarded as the product of the activities of both clasps
of organisms. The average color was 7.3, flavor 3.2, and score
078; the average depreciation from control, color 3.8, flavor 1.7.
and score 234.

Four samples were successfully inoculated witli green mold.
In two instances Penicillium was employed and in the others,
Eurotium. Fluorescent bacteria played a part as associated or-
ganisms in certain of these samples. The average color was 9,
flavor 3.5, and score 600; average depreciation from control.
color 4.8, flavor 2.0, and score 281.

Four sirups were made from saps inoculated with a mixture
of organisms. The composite infection was undertaken to de-
termine the influence of yeasts and molds upon the development
of fluorescent bacteria. The results were not entirely satisfactory

400 Bulletin 167

but indicate that when these two groups of organisms are asso-
ciated in the same sample of sap the fluorescent bacteria are
likely to gain the ascendency. The appearance of the samples
and the results of the plates indicated that the development of
the fluorescent group was stimulated by the presence of yeasts
and molds, with which they must compete. This may account
for the considerable number of failures which resulted from
attempts to inoculate unsterilized sap with cultures of yeasts and
molds. The average figures for the composite samples follow :
Color j.j, flavor 3, and score 663 ; the average depreciation from
control, color 3.3, flavor 1.5, and score 213.

In eight cases, attempts at infection failed or at least the
introduced organism was not recovered. In a majority of these
samples the fluorescent bacteria appeared sooner or later and
exercised an influence upon the quality of the sirup. The average
color was 7, flavor 2.3, and score JJ2 ; average depreciation from
control, color 3.4, flavor 0.3. and score 103.

Three sirups were made from natural sour sap, that is to say.
sap which was allowed to remain in the buckets late in the season
until it had seriously depreciated. One object of this procedure
was to determine what proportion of invert sugar might be ex-
pected in such material. Difficulty is often experienced in graining
sugar made from the last run sirups. This trouble probably
results from a large proportion of invert sugar, which of course
might be formed in the sap by the action of micro-organisms. In
the three sirups here reported, however, the proportion of invert
sugar was low. Two other samples of similar material yielded
sugar which grained readily The average figures for the three
sirups follow: color 20+, flavor 31, and score 333; average de-
preciation calculated on first run control, color 16-}-, flavor I1,
and score 542.

Thirteen samples were reserved for incubator controls. These
were treated in different ways in different series. Some of them
were so handled that they were only slightly inferior to the true
control, while in other instances they were very nearly comparable
to inoculated material. The greater part of the depreciation in

Micro-organisms of M mm, Sap tOI

these samples should be attributed to the fluorescent group of
bacteria. The average color was 5.4, flavor 2, and seme 827;
average depreciation, color 1.8, flavor 0.7, and score 87.

Eleven samples were reserved as controls. Part of these
were held in the cold during the incubation period and undoubt-
edly underwent slight depreciation. The average color was 3.8,
flavor 1.4, and score 911.

Nine samples were made from last run sap obtained from
tap-holes which had become sour, that is from which cloudy sap
was running. The average color was 11, flavor 4.8, and score
397 ; average depreciation from the first run control of the same
reason, color 6.9, flavor 3.1, and score 490.

Nine samples were made from last run material obtained
from the same trees and at the same time as the nine samples
mentioned in the preceding paragraph, but from fresh tap-holes.
It is important to notice that the first three samples of this char-
acter which were made in 1909 possessed a flavor equal to that
of the first run control, and that two of these same sirups were
superior in color to their control. In the remaining six instances,
however, the product of the two succeeding seasons, the sirups
possessed the characteristic buddy flavor and were uniformly 3
grades inferior to the controls. The average color was 5, flavor
3.7, and score 636; average depreciation from first run control
of the same season, color 0.8, flavor 2, and score 243.

If the nine samples of sirups obtained from the sour sap of
the last run arc compared with the nine samples obtained from
the sweet sap of the last run, it is seen that the average deprecia-
tion from souring is 6.1 in color, 1.1 in flavor, and 239 in score.

One sirup was made from sap caught in tin buckets under
cleanly conditions, and one was made from sap obtained the
following days from the same tree but caught in wooden
buckets. This latter sample showed a depreciation of 6 points
in color, 3 in flavor, and 400 in score.

In addition to the groups mentioned above one sirup origin-
ally intended for a control was burned so that it was necessary to
eliminate it from the comparison. The tabulated results follow:

4'»-_' Bulletin 167

TABLE 10. STATISTICAL TABLES SHOWING RELATED SAMPLES IH JUXTAPOSITION

No.

Character of sample

Organism

or organism

number

Count

Reaction

Sucrose

2

Fluorescent,

XXXIII

96.79

102

Ditto,

Ditto,

4.5

95.67

O

Ditto.

XXXVI

96.73

L03

Ditto,

Ditto.

1.4

95.38

4

Ditto,

L

96.54

5

Ditto,

LI 1 1

97.16

7

Ditto,

LVI

96.16

8

Ditto,

5

97.14

101

Ditto,

5

L.2

96.45

I'll

Ditto,

(XXXIX

L2350000

94.91

30

Ditto,

CLIX

3262

93.75

11

Ditto,

CLXVII1

90 100

IO.11

93.03

LOO

Ditto,

Ditto,

1.1

96 21

69

Ditto,

CLIV

195 mi

10.0

93.87

70

Ditto,

(XXXIII

20400000

10.0

93.47

71

Ditto,

CXLVII

234001

6.5

93.69

72

Ditto,

CLIII

29250000

10.0

95.07

73

Ditto.

CLVIII

123400000

2.3

97.47

91

Ditto,

CXL

1.5

94.76

92

Ditto,

CXII

1.4

95.00

it:;

Ditto,

LI

1.2

95.43

94

Ditto.
Average,

CXLI

1.2

94.6S

9

Non-fluorescent,

XXVI

95.61

10

Ditto,

XXX

95.86

12

Ditto,

I

95.71

55

Ditto,
Average,

CVII I

2730000

6.0

94.76

75

Spore-bearer plus*

CLXXIX

10400000

10.0

84.74

76

Ditto,

CLV

1875000

10.7

87.03

77

Ditto,

CLXXVII

11700000

10.1

88.13

78

Ditto,

XXXVI

14625000

11.2

83.86

79

Ditto,

CLXIII

6500000

:':..l

78.78

80

Ditto,

CLXXVII I

9750000

12.5

79.20

81

Ditto,
Average

3250000

10.0

11

B. aceris,

LXXXVII

170.0

96.12

95

Ditto,

Ditto,

27.0

90.85

90

Ditto,
Average,

CCXVII

25.4

86.72

*I. e., spore-bearing organisms associated with fluorescent and other

organisms. (See pages 375 to :!77. )

Micro-organisms of M \pi.i- Sap 103

l LBLE 10. STATISTN A I. I ABLES SHOWING BELATED SAMPLES IN Jl XTAPOSITION

Ratio

Color

Flavor

Score

in PR] (1 vi [ON

Inv. Sug.

Color

Flavor

Score

Date

1.55

62

14

4

425

8

9
.J

450

3-2 1 09

1.1 1

M

7

4

600

1

9

350

3-26-1 1

2.09

46

11

2

700

5

1

175

3-24-09

1.35

71

7

4

600

4

•>
>>

350

3-26-1 1

1.56

62

9

2

7 .Mi

■ »

1

125

3-2 1 09

1.44

67

11

2

Tun

5

1

L75

••

1.G4

59

10

2

725

4

1

L50

3-27-09

1.15

84

Id

2

725

4

1

150

■ .

0.94

103

8

■ 1

700

5

2

250

3-26-11

0.38

250

8

2'

750

4

ii'

L25

3- 7-10

0.89

105

8

2

775

4

0

100

1.40

67

9

675

5

2

250

3-14-10

0.93

103

—

0

725

4

2

225

3-26-11

1.59

59

9

6

675

5

2

250

3-22-Ki

2.46

38

11

4

500

7

•>

425

..

0.58

162

14

4

425

10

3

500

ii

0.87

109

13

4

450

9

n

■ >

475

"

0.68

143

6

2

825

2

l

100

1 1

0.40

237

6

•>

750

o

l

150

3-22-1 1

0.52

183

7

:;

725

4

l

175

0.43

222

6

2

825

o

O

0

75

t <

0.52

182

7

4

600

4

2

300

"

9

2.9

665

4.S

1.6

242

0.78

122

5

2

850

— 1

1

25

3-27-09

0.77

124

6

750

0

2

125

2.23

43

6

•>

o

750

0

2

L25

it

2.38

40

5

2

850

3

1

L25

3-20-10

5.5

2.5

800

0.5

1.5

100

Ki.77

8

9

o

675

5

2

250

3-24-10

8.94

10

10

■ »
•J

650

6

2

275

«

8.24

11

8

3

700

4

2

225

10.45

8

8

4

575

4

•>

350

..

15.25

5

7

4

600

0

3

325

16.13

5

10

4

525

6

- >

40(i

"

7.51

12

7

4

600

•>

3

325

a

8.4

3.6

618

4.4

2.6

307

1.72

56

7

4

600

1

9

•J

275

3-27-09

5.04

18

6

•i

• >

750

■>
O

1

150

3-22-11

7.20

12

7

4

600

4

2

300

••

6.7

3.7

650

2.7

2

242

4t>4

Bulletin 167
table 10- -Continued

No.

Character of sample

Organism

or organism

number

Count

Reaction

Sucrose

4G

Pink coccus,

CIV

11300000

10.0

95.07

47

Ditto,

IV

1950000

9.6

88.81

50

Ditto,

XLIX

1950000

8.0

93.83

51

Ditto,

CVI

4225000

3.5

97.30

107

Ditto,

Ditto,

4.0

94.52

54

Ditto,
Average,

CXXVI

1300000

8.5

94.11

98

Pink yeast,

CXXXII

1.5

86.70

14

Red yeast,

25

9.0

95.30

62

Ditto,

Ditto,

650000

9.5

89.27

15

Ditto.

LXII

5.0

93.26

114

Ditto,

Ditto,

5.0

96.31

18

Ditto,

LXXIX

12.0

91.65

35

Ditto,

CXI

2255000

95.13

42

Ditto,

CVI I

13650000

19.0

93.27

59

Ditto,

CXXIII

325000

8.0

94.74

GO

Ditto,

CXIII

1950000

8.4

95.44

01

Ditto,

CX

650000

5.5

95.80

99

Ditto,

Ditto,

1.4

94.82

63

Ditto,

CXIV

975000

11.0

89.06

04

Ditto,
Average,

CXX

97500

10.0

91.64

13

Gray yeast.

24

93.34

28

Ditto,

Ditto.

437000

82.83

109

Ditto,

Ditto.

41.0

67.33

48

Ditto.

CLXXX

1625000

16.7

93.89

52

Ditto,

CLXII

1275000

6.0

96.84

53

Ditto,

CLXXI

630000

6.0

92.13

110

Ditto,

CXXI

3.3

97.09

115

Ditto,
Average,

Ditto

4.7

95.14

16

Green mold.

LXIV

8.2

95.73

17

Ditto,

LXV

8.0

92. 7f,

111

Ditto,

CCCI

2.0

93.:n

117

Ditto,
Average,

Ditto,

7.4

91.09

19

Composite,

5.0

93.28

116

Ditto,

3.7

95.01'

118

Ditto,

4.8

95.69

119

Ditto,
Average,

4.2

96.27

Micro-organisms of Maim,!'. Sap t(|">

fable in— Continued

Ratio

Color

Flavor

Score

DEPRECIATION

Inv. Sug.

Color

Flavor

Score

Date

1.05

91

6

2

NLT>

2

1

100

3-14-10

7.90

11

6

Q

750

2

2

ITT,

CI

2.20

43

6

21

800

4

r

17.".

3-20-10

0.G5

150

4

2

875

2

l

100

C<

0.G9

137

5

4

650

2

2

250

3-27-11

2.71

35

7

2=

750

5

1-'

225

3-20-10

5.7

2.6

775

2.8

1.6

171

8.G9

10

9

4

550

6

400

3-26-11

3.01

32

10

2

725

4

1

150

4- 1-09

7.30

12

9

4

550

6

3

400

3-21-10

2.98

31

8

2

775

2

1

100

4- 1-09

1.01

95

5

O

775

1

1

100

3-30-11

3.12

29

10

725

4

1

150

4- 1-09

1.15

83

12

21

650

8

01

225

3- 7-10

2.14

44

5

22

800

1

r

125

3-14-10

2.03

47

7

3

725

4

2

225

3-21-10

1.76

54

6

O

O

750

3

2

200

it

1.77

54

9

675

6

2

275

ti

2.05

46

7

4

600

4

3

350

3-26-11

7.63

12

8

3

700

5

2

250

3-21-10

5.86

16

8

3

700

5

2

250

CI

8

2.8

703

4.1

1.7

215

2.53

37

7

2

800

1

1

75

4- 1-09

12.15

7

11

4

500

7

2

375

3- 7-10

28.35

2

10

4

525

7

2

375

3-27-11

2.35

40

6

2=

775

2

1-

150

3-14-10

1.21

80

5

2

850

o

1

125

3-20-10

3.96

23

6

3

750

4

2

225

it

1.74

56

6

4

625

O

2

275

3-27-11

1.52

63

7

4

600

3

2

275

3-30-11

7.3

3.2

678

3.8

1.7

234

2.61

37

10

4

525

4

3

350

4- 1-09

3.47

27

10

2

725

4

1

150

it

4.39

21

7

4

600

4

2

300

3-27-11

3.72

25

9

4

550

5

2

325

3-30-11

9

3.5

600

4.3

2

281

4.09

23

9

3

675

3

2

200

4- 1-09

0.68

141

8

4

575

4

2

300

3-30-1 1

0.98

98

7

4

600

O
O

2

275

a

0.81

119

7

2

800

3

0

75

1 1

7.7

o
t>

663

3.3

1.5

213

4-1 m;

Bulletin 167

tahle 10 — Continued

No.

Character of sample

Organism

or organism

number

Count

Reaction

Sucrose

31

Failure,

:,';r,7000

96.G2

32

Ditto,

r.iiuOOOO

95.91

DO
£>0

Ditto,

1950000

95.G1

36

Ditto,

7::i2

9G.1G

37

Ditto,

8825000

95.88

39

Ditto,

9750

9G.18

L06

Ditto,

4.1

97.30

108

Ditto.
Average,

3.3

95.37

84

Sour sap kept.

91.14

85

Ditto,

11275000

CO

94.68

86

Ditto,
Average,

11275000

6.0

93.21

Depreciation on

foregoing cal

culated on fir

st run control.

1

Incubator control.

96.84

34

Ditto,

7312000

95.86

38

Ditto,

162500

94.80

40

Ditto,

5850000

95.05

43

Ditto.

1625000

6.5

94.19

45

Ditto.

19500

4.5

9G.28

56

Ditto,

320000

3.1

96.02

."

Ditto,

37000

2.5

96.47

65

Ditto.

L950000

9.0

91.89

96

Ditto.

0.5

95.33

mi

Ditto,

0.6

97.00

112

Ditto.

1.0

97.i-.ii

120

Ditto,

Average.

6.4

95.02

6

Control.

95.41

20

Ditto,

2.0

95.37

27

Ditto,

10000

96.19

41

Ditto,

66

1.2

97.82

49

Ditto.

100

1.0

96.51

58

Ditto,

380

3.0

97.01

68

Ditto,

500

2.5

96.90

89

Ditto,

1.2

95.81

97

Ditto,

0.5

96.04

L05

Ditto,

1.0

96.23

1 i:;

Ditto,
Average,

2.0

96.97

Micro-organisms oi' Maple Sap 401

table LO — Continued

Ratio

Color

Flavor

Score

in i'i:i < i vi ion

Inv. Sug.

Color

Flavor

Score

Dah'

0.30

322

6

2

825

2

0

50

:;- 7 L0

0.40

208

6

2

825

2

0

50

• •

0.23

416

7

2

800

•>

0

75

..

0.6]

1 r,s

9

21

725

5

0l

l.-,o

"

0.64

150

11

21 '

675

7

II'

200

"

0.26

370

7

2

800

3

0

7.'.

4 (

0.66

147

7

•>
o

725

4

1

175

3-27-1]

0.90

LOG

4

o

800

1

1

L00

CI

7

2.:\

772

3.4

• >

L03

2.83

32

20

01

U

350

16

r

525

4- 2-10

L.57

60

20+

•11
Q

325

16+

i1

550

..

L.23

76

20 +

•M

32:,

16+

r

550

• •

20

31

ooo

16

r

542

1.07

90

7

1

850

1

0

25

3-24-09

0.42

228

6

21

800

2

01

75

3- 7-la

0.48

198

5

vi

825

1

0l

50

"

0.64

149

9

2

750

5

0

125

. .

2.64

36

n
1

2

800

o

o

1

L25

3-14-10

0.51

189

4

1

925

0

0

0

"

1.35

71

5

2

850

• »

1

125

3-20-10

0.60

161

2

1

975

0

0

0

a

3.52

26

6

4

625

■>
o

• >

325

3-21-10

0.44

217

5

2

850

2

0

50

3-22-1]

0.41

237

4

2

875

1

1

75

3-20-1 l

1.11

88

5

2

850

2

0

50

3-27-11

0.57

107

5

9
O

775

1

1

100

3-30-11

5.4

2

827

1.8

.7

87

0.97

98

6

1

875

3-24-09

1.58

60

6

1

ST.".

4- 1-09

0.30

321

1

2

875

3- 7-lo

0.45

217

1

1

92T.

3-1 llo

0.42

230

2

1

975

3-20-10

0.43

226

•>

1

950

3-21-10

0.51

190

1

1

925

3-22-10

0.34

282

2

900

3-22-11

0.19

505

O

•>

1

950

3-26-11

0.24

403

3

2

900

3-27-1]

0.61

159

4
3.8

2
1.4

S75
911

3-30-11

408 Bulletin 167

table 10 — Concluded

No.

Character of sample Organism

or

organism number Count

Reaction

Sucrose

22

Last

run sour,

95.27

23

Ditto,

92.56

25

Ditto,

95.05

88

Ditto,

73125000

4.0

94.56

121

Ditto,

93.94

123

Ditto,

95.33

125

Ditto,

93.88

127

Ditto,

87.41

130

Ditto,

Aver,

age,

93.86

Depreciation on foregoing calculated on first

run control.

21

Last run sweet,

95.19

24

Ditto,

96.02

26

Ditto,

96.42

87

Ditto,

5

2.5

97.27

122

Ditto,

95.80

124

Ditto,

98.54

12G

Ditto,

94.46

128

Ditto,

94.21

129

Ditto,

95.34

Average,

Dej

preciation on foregoing calculated on first

run control.

22

Last run sour,

95.27

21

Ditto,

sweet,

95.19

23

Ditto,

sour.

92.56

24

Ditto,

sweet,

96.02

25

Ditto,

sour,

94.05

26

Ditto,

sweet,

96.42

88

Ditto,

sour, 73125000

4.0

94.56

87

Ditto,

sweet, 5

2.5

97.27

121

Ditto,

sour,

93.94

122

Ditto,

sweet,

95.80

123

Ditto,

sour,

95.33

124

Ditto,

sweei .

98.54

125

Ditto,

sour,

93.88

126

Ditto,

sweet,

94.46

127

Ditto,

sour.

87.41

128

Ditto,

sweet,

94.21

130

Ditto,

sour,

93.S6

129

Ditto,
Avera

sweet,
ge depreciation of sour samples,

95.34

66

Tin bl

icket, 140

2.0

96.62

67

Wood

bucket, 6500000

5.5

88.25

74

Burned

1 control, 750

3.0

96.36

Micro-organisms of .\l\ru-: Sap 409

TABLE 10 — COHclinlfii

Ratio

Color

Flavor

Score

DEPREI 1 \ HON

Inv. Sug.

Color

Flavor

Score

Date

2.76

35

8

o
o

700

2

2

175

4-12-09

3.55

26

14

3

550

8

2

325

"

3.13

30

14

3

550

8

2

325

4-16-09

1.79

53

11

6

250

7

4

625

4- 3-10

0.34

276

7

5

450

4

3

15(1

4-21-11

0.G4

149

11

6

250

8

4

650

4-24-11

0.53

125

7

5

450

4

O

o

450

a

4.59

19

15

6

150

12

4

750

4-27-11

1.34

7(1

12

6

225

9

4

675

4-28-11

11

4.8

397

6.9

3.1

492

0.82

116

3

1

950

-3

0

-75

4-12-09

0.61

157

1

850

1

0

25

a

0.51

189

5

1

900

-1

0

-25

4-16-09

0.26 '

374

4

5

525

0

o

L'5(i

4- 3-10

0.67

143

5

5

500

2

o
o

400

4-21-11

0.59

167

5

5

500

2

o

■ »

400

4-24-11

0.31

305

6

5

475

3

3

425

a

0.31

304

4

5

525

1

o
o

1175

4-27-11

0.12

794

5

5

500

2

3

400

4-28-11

5

3.7

636

.8

2

243

2.76

35

8

O

700

5

2

250

4-12-09

0.82

116

o

1

950

' I

3.55

26

14

o
o

550

7

2

300

it

0.61

157

7

1

850

tt

3.13

30

14

o

550

9

2

350

4-16-09

0.51

189

5

1

900

it

1.79

53

11

6

250

7

1

275

4- 3-10

0.62

374

4

5

525

it

0.34

276

7

5

450

2

0

50

4-21-11

0.67

143

5

5

500

a

0.64

149

11

6

250

6

1

250

4-24-11

0.59

167

5

5

500

If

0.53

125

7

5

450

1

0

25

((

0.31

305

6

5

475

II

4.59

19

15

6

150

II

1

375

4-27-11

0.31

304

4

5

525

<(

1.34

794

12

G

225

7

1

275

4-28-1 1

0.12

70

5

5

500

0.1

1.1

239

<i

0.41

236

O

1

950

3-22-Ki

6.91

13

9

4

550

6

3

400

3-24-10

0.70

138

9

4

550

5

6

375

3-24-10

410

Bulletin 167

TABLE 11. SUMMARY OF AVERAGES OF RELATED GROUPS IN ORDER OF SCORE

No.

Croup

Number
of samples
in group

Sucrose

1

Till buckets,

1

96.62

2

Controls,

11

96.32

Incubator controls.

13

95.55

4

Non-fluorescent bacteria,

4

95.50

• >

Pink cocci.

G

93.94

•

<;

Failures,

8

96.13

7

Red yeasts,

13

93.54

8

Gray yeasts.

8

89.72

fi

Fluorescent bacteria,

22

95.42

L0

Composites.

4

95.22

11

B. aceris,

:;

91.16

12

Last run sweet.

9

95.92

l;;

Fluorescein and spore-bearing bacteria,

7

84.42

14

Green molds.

4

93.21

15

Pink yeast,

1

86.70

1G

Wooden buckets

1

88.25

17

Burned control.

1

96.36

IN

Last run. sour,

9

93.41

L9

Sour sap kept,

3

93.00

Micro-organisms oi? Maple Sap III

rABLE II. sIMMAKV OF AVERAGES Ol RELATED GROUPS l\ ORDER OF SC0R1

HI l'l:i < 1 VI [OB

Inv.

Sug.

Ratio

Color

Flavor

Score

Color

Flavor

Score

0.41

236

3.0

1.0

950

0.60

161

3.8

1.1

911

1.04

90

5.4

2.0

827

1.8

0.7

NT

1.52

62

5 . 5

2.5

800

0.5

L.5

1 Mil

2.45

37

5.7

2.6

77:.

2.8

1.6

171

0.5]

74

T.il

2.3

771'

3.4

0.3

in:;

3.18

29

8.0

2.8

703

1.1

1.7

2 1 5

G.86

L3

7.:;

3.2

678

3.8

1.7

23 1

1.09

88

9.0

2.9

665

4.8

L.6

242

1.64

58

7.7

3.0

663

1.5

213

4.70

20

6.7

3.7

650

2.7

2.0

2 12

0.47

.'ill

5.0

3.7

636

0.8

2.0

243

11.03

8

8.4

3.6

618

4.4

2.6

307

3.56

26

9.0

3.5

600

4.3

2.0

281

8.69

10

9.0

4.0

550

6.0

3.0

100

6.91

13

9.0

4.0

550

6.0

3.0

too

0.70

138

9.0

4.0

550

6.0

3.0

lllll

2.07

45

11.0

1.8

397

6.9

:;.i

492

L.88

49

20.0

.>■>..

L6.0

l1.

542

412 Bulletin 167

comparison oe sirups made from inoculated sap and from

natural sour sap

The reader with a practical turn of mind may well inquire
how the quality of the sirups made from the inoculated saps
compares with that of the sirups made from saps which have be-
come sour from natural causes. It might seem that the artificial
introduction of large numbers of bacteria into sap would result in
the production of an abnormal sirup, far inferior to that secured
from saps souring under natural, and perhaps unavoidable, con-
ditions ; but such is not the fact. Much sirup and sugar are
annually made from sap more seriously injured by natural
souring than was most of that discussed in the foregoing
pages. The organisms at work are the same in either case,
but the conditions under which souring occurs in nature are
as a rule more favorable to bacterial growth than were those
maintained in the incubator. In nature, however, there is al-
most always a mixed infection, in which the predominant forms
are those most seriously injurying the color. In the incu-
bator it was possible so to control conditions as to give the
advantage to particular groups at will, so that in some cases the
flavor of the sirup was more seriously injured than is likely to
occur in nature unless the conditions are extremely bad. The
writer, while unwilling to acknowledge himself a great offender,
has personally made sirups for commercial sale from saps in-
jured more seriously by natural souring than were most of those
employed in these experiments. In fact only the most pains-
taking care can prevent the occurrence of such injury towards
the close of the season.

The facts are brought out clearly by a study of the data
already presented. Twelve of the sirups discussed were made
from sap which had soured naturally. The average color was
8.3, flavor 4.3, and score 381 ; average depreciation from first run
control, color 9.2, flavor 2.6, and score 504. Three of these
samples, however, (Nos. 84. 85 and 86) were made from sour sap
which had been allowed to stand two or three days in the buckets
so that it was very poor indeed: and six (Nos. 88, 121, 123, 125.

Micro-organisms oi? Maple Sac H3

127 and 130) were made from sour sap drawn very late in the
season, so that it was not only sour but buddy. The three re-
maining samples (Nos. 22, 23 and 25), however, were made from
sour sap free from buddy flavor. Samples 22 and 25 were con
centrated to sirup the same day they flowed, and at the time of
evaporation the first drops to run had not hcen out of the \
cular tissue more than twelve hours. Sample 23 was evaporated
thirty-six hours after it began to run. These sirups might he ex-
pected to be at least as good as the ordinary material made from
sour sap and probably superior to it. They are therefore used in
the following comparison of depreciation values, which values in
these cases are calculated from the average of all controls of the
three seasons, instead of from the average of first run controls.
The average color was 12, flavor 3, and score 600; average de-
preciation from all controls, color 8.2, flavor 1.6, and score 311.

DISCUSSION OF DEPRECIATION VALUES

The specific influence of the various groups of organisms
and of natural souring upon sirup is seen most clearly from a
study of the depreciation values. If the groups are arranged ac-
cording to the average amounts of depreciation in color they fall
into the following order :

TABLE 12. ORDER OF

AVEBAGE DEPRECIATION IN COLOB

Depreciation

Group

Color

Flavor

Score

1

Non-fluorescent bacteria,

(».:»

1.:.

100

2

Incubator control,

1.8

0.7

87

0

0

B. aceris.

2.7

2.0

242

4

Pink coccus,

2.8

1.6

171

5

Composites,

3.8

1.5

213

6

Failures,

3.1

0.3

103

7

Gray yeast,

3.8

1.7

234

8

Red yeast,

4.1

1.7

215

9

Green mold.

1.3

2.0

281

10

Fluorescent bacteria plus

spore

■bearers, 1.1

2.6

307

11

Fluorescent bacteria.

l.s

1.6

242

12

Natural sour sap,

8.2

1.6

311

A glance at the foot of the table shows that the sirups mosl
seriously injured in color were made from sap souring naturally.

II I

i'.ru.KTix H>7

The sirups showing the next most serious depreciation in color
were those inoculated with green fluorescent hacteria. Then
comes a group in which fluorescent organisms were employed in
the inoculation and developed in association with such spore-
bearers as were present. It has already been shown that the
fluorescent bacteria exercised some influence on the sirups of the
next five groups also, while in the remaining groups they were
either absent or present in smaller numbers for a shorter time.
If the depreciation figures are arranged according to flavor
values a different order is secured, as follow-:

TABLE 13. ORDEB OF AVERAGE DE1*RE< l.\TI<>\ IX FLAVOR

Group

1 Failures,

2 [ncubator control.

3 Xon-fiuorescent bacteria,

4 Composites,

5 Pink coccus,

6 Fluorescent bacteria,
? Natural sour sap.
8 Gray yeast,
!' Red yeast,

10 B. aceris,

11 Green molds.

12 Fluorescent bacteria plus sporedjearers. 4.4

Depreciation

olor

Flavor

Sco

3.4

0.3

m:;

1.8

0.7

s7

0.5

i.:»

100

3.3

1..-,

213

1.6

171

4.8

1.0

2 4 2

8.2

/.<;

.;//

3.8

1.7

23 i

4.1

1.7

215

2.:

2.0

242

4.:!

2.0

2S1

301

The spore-bearing bacteria, green molds. B. aceris, both
groups of yeasts and the natural sour material, occupy positions in
this table below the fluorescent group. The sirups of five of these
six groups were influenced somewhat in quality by the fluores-
cent bacteria, and it is probable that the contrasts in quality are
not so great as they would have been had the fluorescent bacteria
been entirely eliminated ; yet it still appears perfectly clear that
tlie organisms which most seriousl) injure the flavor ^^i maple
sirup are not those producing the darker grades of color.

If the groups are re-arranged in the order of depreciation in
-core, an idea may be gained of their relative commercial values.

Micro-organisms of Maple Sap 1 1 5

TABLE 14. OBDEB 01 AVERAGE DEPRECIATION l\ SCOB1

Depreciation

Group

Color

Flavor

Score

1

Incubator control.

1.8

0.7

^7

•>

Non-fluorescent bacteria,

0.5

1.5

1011

3

Failures,

3.4

0.3

103

4

Pink coccus.

2.8

L.6

171

5

Composites,

3.3

1.5

213

6

Red yeast,

4.1

1.7

215

7

Gray yeast.

3.8

1.7

•23 1

8

Fluorescent bacteria,

4.8

1.6

242

9

B. aceris,

2.7

2.0

l»Il»

10

Green molds,

4.;:

2.0

281

11

Fluorescein bacteria plus

spore-bearers, 4.4

2.6

307

t2

Natural sour sap.

' j ...

i.i;

.;//

DISCUSSION Hi- LAST RUN SIRUPS

Examination of the statistical tables shows that the dark
color of late run sirup is to be attributed to the action of micro-
organisms, since the sirups made from last run material gathered
without infection were always of a light hue. The average de-
preciation in color as calculated upon the first run controls was
0.8 point. At first sight it would appear that the results as ap-
plied to flavors lack uniformity. The sirups of the first sea- m
suffered no depreciation in flavor while those of the last two
seasons uniformly fell off three points; but, as has been already
suggested, this result is accounted for by the meteorological di-
vergencies of the several seasons (pages 328 and 380).

IXFLUKXCE OF INERT EXTRANEOUS MATERIAL

Before formulating answers to the questions propounded at
the outset, attention is called to the fact that in these studies no
attention has been paid to the influence upon the color and flavor
of sirup of inert extraneous matter, such as colored rain water,
bark, insects, caramel and the like. Everyone recognizes the
importance of eliminating such material, but the following para-
graph showing the intluence of caramel may be worthy of record.

The commercial supply of sap from the sugar place where
the inoculation experiments were carried out was concentrated

416 Bulletin 167

in a multiple pan evaporator in which the sirup was always drawn
off from the rear pan. Of course the usual accumulation of the
so-called niter (mainly malate of lime) occurred in the sirup
pan. During the height of the season the capacity of the equip-
ment was hardly sufficient for the number of buckets hung, so
that in order to save time on one occasion the daily cleansing of
the pan was omitted. A decided darkening was soon noted in the
grade of sirup obtained and suspicion was directed towards the
sirup pan. The fire was banked and the niter removed. An im-
provement of several points in color of the sirup immediatelv
resulted, showing that the sugar mechanically included in the
niter was being caramelized and was exercising a detrimental
influence. After this the color of the sirup was carefullv
watched, and whenever a depreciation in color began to appear
the niter was removed from the sirup pan and the former light
light color of the sirup was restored.

Conclusions

Returning now to the questions these studies were inaugu-
rated to answer, the work has shown that the depreciation in color
and flavor of maple sirup which ordinarily occurs in the com-
mercial sugar place as the season progresses is to be attributed t< 1
the action of micro-organisms. Certain groups exercise a more
detrimental influence upon color than upon flavor, while with other
groups the reverse is true. The influence of each group appears
more or less specific and characteristic. The most common form
of organisms present in maple sap, the fluorescent bacteria, in-
jures the flavor much less than it does the color. Those organ-
isms which most seriously affect the flavor of sirup, the non-
fluorescent, spore-bearing bacteria, molds, and stringy sap organ-
isms, do not seriously darken the color. They do. however, fre-
quently render the sirup cloudy and so viscid that it does not clear
perfectly, even if left undisturbed for months.

In addition to the depreciation due to bacteria there may
occur deterioration due to physiological changes in the tree itself.

-

-

X

I

::

a

z

o

_ I-

_ r-

- --

c

— u
« «2

- '-

< E

>. -
x —

- -
_ >

_ J

M*

c n

~ -

C "r

C

- _

Micro-organisms of Maple Sap 417

In certain seasons these changes which produce the "buddy"
flavor may not occur in Vermont until after the sap (low ceases.
In ordinary years, however, the season is interrupted by periods
of growing- weather, so that the vegetative activity of the tree is
resumed some time before the final discontinuance of night
freezes, and the influence of these physiological changes becomes
manifest in the true "buddy" flavor which appears in the sirup.
Formerly the opinion was commonly held that this depreciation
was due to the presence of a relatively large proportion of invert
sugar. An examination of the analytical data clearly demon-
strates that this view is erroneous. There is a decline rather
than an increase in the content of invert sugar as the season
advances. The exact nature of the cause of the true buddy flavor
is unknown. In this connection, attention is ag'ain directed to
the statement on page 346, to the effect that frequently the s< ■-
called buddy material is not buddy in the sense in which the term
is employed in this bulletin. Much sirup popularly but errone-
ously termed buddy possesses objectionable foreign flavors due
solely to the action of micro-organisms.

REMEDIAL MEASURES

Practical remedial measures must be based upon efforts to
minimize the contamination with micro-organisms and to restrict
the period of their action to the shortest possible time. The
lower their content and the shorter their period of growth, the
better the product. As in dairying, cleanliness must be the watch-
word of the producer of superior goods. Clean sponts, clean and
covered buckets, and clean holders are necessities. The use of
metal utensils is to be preferred to the employment of wooden
ones, because the latter material affords organic matter upon
which organisms may develop. Moreover wooden utensils are
less readily cleaned. Covered buckets are preferable to open ones,
not only because they keep out rain and- snow, but because they
prevent the entrance of bits of falling bark, decayed wood, and
other inert matter. Such material is always heavily charged

418 Bulletin 167

with bacteria and other organisms, so that in addition to the
coloring matter carried in the refuse itself, agents are introduced
which further discolor the product through their vital activities.

The practice of storing sap is one to be avoided whenever
possible. Modern evaporators not only make long periods of
boiling unnecessary, but they make it possible to concentrate the
runs day by day as they occur. They are doubtless important
factors contributing to the improved quality of evaporator sirup
as compared with that produced by older methods. When storage
is resorted to, the temperature of the tank should be kept as low
as possible, because the lower the temperature the slower the
micro-organic development. Holders should be located without
rather than within the boiling house, where the heat of the pans
will not influence their temperature.

The evidence obtained indicates that the sugar-maker cannot
expect to produce a high quality of sirup at the close of the season
in average years , because there is no known means by which the
physiologically induced "buddy" flavor may be avoided. So long
as the depreciation is caused solely by bacteria, cleanly methods
will enable the producer to maintain a high standard of excellency
in his product ; but if the physiological activity of the tree begins
to be manifest, the producer will find himself unable to manufac-
ture an article of high excellency as regards flavor. The light
color can be maintained indefinitely, but the "buddy" flavor is
so objectionable that the market value of the sirup is insufficient
to render its production profitable.

Micro-organisms op Maple Sap 419

PART II

DISCUSSION OF PHYSICAL AND CHEMICAL DATA SECURED ON

MAPLE SI HIPS OBTAINED FROM SAPS INOCULATED

WITH MICRO-ORGANISMS.

By C. H. Jones

The analyses of maple sirup displayed in tables 15-37
(pages 420 to 457) were made on samples secured in the prosecu-
tion of the work discussed in the preceding pages. They were
obtained during three successive sugar seasons and include not
only pure sirups made under the most favorable conditions pos-
sible, but, also, those representing the extremes of both nat-
ural and artificial inoculation with bacteria, yeasts and molds.

The samples were sterilized at the time of manufacture and
stored in the dark in sealed half-pint ''lightning" jars. All
analyses were made immediately after opening the jars. Particular
attention was paid to the sucrose and invert sugar contents, but,
in addition, the moisture, ash, and malic acid value were secured
in order to determine the effect of the treatment on the data
usually employed in judging the purity of maple products. The
analytical methods employed were those in ordinary use, as out-
lined in bulletin 134 of the Bureau of Chemistry of the United
States Department of Agriculture.

It should be remembered that these samples were all pure
maple sirups so far as admixture with cane or other sugars is
considered. They were, however, either naturally or artificially
inoculated with the types of micro-organisms normally present
in maple sap. This inoculation, as will be shown, has a greater
influence on color and flavor (physical characteristics) than on
the chemical composition.

A total of 128 samples were examined. The analytical re-
sults are reported in groups, based mainly on the nature of the
inoculating organisms employed. The sample numbers and
groupings correspond to those outlined on pages 402 to 409.

42< I

r.iu.ETiN [67

Before beginning a detailed discussion, attention should be
called to a few general conditions obtaining in the manufacture
and analysis of the sirups, which doubtless to some extent
affected the chemical data.

1. All samples were secured from the same sugar orchard.

2. They were made during three successive sugar seasons.

3. The size of the sample and the manner of its evapora-
tion made it difficult closely to control the end boiling point and.
consequently, large individual and yearly variations were observed
in the concentration of the sirups, as is shown below.

TABLE 15. MOISTUKE CONTENTS OF SIBUP SAMPLES

Number of

Extreme

samples

yearly

secured

Year

Average

Maximum

Minimum

variation

%

'<

%

''<

26

1909

33.67

38.ss

29.44

9.44

GO

1910

38.01

47.60

32.46

15.14

42

1911

30.39

38.97

26.76

12.21

The average obtained in 1909 more nearly approaches the
eleven-pound per gallon standard,1 which calls for a moisture
content of from 34 to 35%, than do those secured in the two fol-
lowing years. The 1910 samples were nearly all light weight
goods, with an extreme variation of 15.14$ in moisture. The
average for 191 1 indicates that most of the samples were con-
siderably more concentrated than the eleven-pound standard1 re-
quires. A thermometer was used to determine the density during
manufacture, but, unfortunately, due allowance was not made for
the boiling points of different grades of sirup. These figures will
be referred to later during the discussion of the ash data (pages
464 and 465).

No particular care was taken to remove the "niter" in the
laboratory previous to the analysis of the 1909 samples, Nos. r-26.
It was thoroughly removed from the 102 samples of the two
succeeding seasons by sedimentation during a period of seven
months.

'Vt. Sta. Bui. 26 (1891); Rut. 18, p. 334 (1905); U. S. Dept. Agr..
Bu. Chem., Bui. 134, pp. 74-75 (1910).

Micro-organisms oi? Maple Sap l_M

EXPLANATION OE PHYSICAL AND CHEMICAL DATA SHOWN IN

TABLES 1 6 TO 34

The chemical analysis of each sample was preceded by
a careful physical examination, for the details of which see pages
345 to 349. The color was determined after the standard of
Bryan1 which comprises twenty grades. (See page 349). The
first grade is never obtained in practical manufacture. As com-
mercially rated for average crops, any grade helow 8 may be
considered No. 1, from 8 to 11 inclusive No. 2, and from 12
to 15 inclusive No. 3.

The sirups were grouped in 6 classes as regards flavor.
No. 1 corresponding- to prime, 2 good. 3 medium, 4 poor, 5
buddy, and 6 buddy and rank. A certain few sirups grading
good and medium, were designated 21 and 22, and similarly a
few, falling between medium and poor, were rated 31. A flavor
of 5 or 6 would immediately condemn the sample for commer-
cial purposes.

The scoring" system adopted for these sirups is based on
the flavor and color as explained on pages 346 to 349.

The column headed "undetermined" is obtained by sub-
tracting the sum of the moisture, sucrose, invert sugar, ash
and malic acid value from 100. It is, of course, affected by
all the errors in the five determinations mentioned, and includes
proteids, tannin, organic acids and other non-sugars.

Group i. Controls. As the word "control" indicates, these
^samples were prepared from sap selected to serve as checks
on experimental procedure. The first two lots, Nos. 6 and 20,
were not boiled fresh, but were held in the cold for about
3 days during the incubation period. All the other saps in this
group were strictly fresh when boiled.

The resulting sirups were of fine flavor and light in color.
No. 49 is credited with the highest score possible, 975, while
5 more of the 11 lots are hardly inferior to No. 49. They
would all be classed as of the highest grade.

'U. S. Dept. Agr., Bu. Chem., Bui. 134, p. 15 (1910).

422

BUIXKTIN 167

TABLE 16. MAPLE SIRUP. CONTKOLS

•—

CD
JO

a

3

a

o>

3

to

.a

m

J3
0>

3
>

■a

O

a

a

•a

a

0

U

0

>

.2

•

0

3

<D
09

O

O

3

w

>

a

cd

O

E-i

0
0

3
O

SQ

a
»— 1

rt

a

%

of

/O

%

%

%

%

%

%

6

6

1

875

33.99

62.98

0.64

0.62

0.27

0.35

0.61

1.16

20

G

1

875

33.54

63.38

1.05

0.83

0.25

0.58

1.04

0.16

27

4

2

875

34.46

63.04

0.20

0.49

0.34

0.15

0.48

1 QQ

l.OO

41

4

1

925

39.78

58.46

0.62

0.46

0.22

0.24

0.53

0.15

40

2

1

975

38.39

59.46

0.26

0.50

0.24

0.26

0.51

0.88

58

0
0

1

950

36.30

61.80

0.28

0.47

0.28

0.19

0.53

0.62

68

4

1

925

37.33

60.73

0.32

0.42

0.28

0.14

0.51

0.69

89

0

2

900

29.96

67.11

0.24

0.43

0.22

0.21

0.73

1.53

97

O

1

950

30.36

66.88

0.13

0.47

0.30

0.17

0.51

1.65

105

2

900

30.53

66.85

0.17

0.55

0.38

0.17

0.42

1.48

113

4

2

875

29.99

67.89

0.43

0.53

0.35

0.18

0.51

0.65

Averagi

-, 3.8

1.4

911

34.06

63.51

0.39

0.52

0.28

0.24

0.57

0.95

Max.,

6

2

975

39.78

67.89

1.05

0.83

0.38

0.58

1.04

1.65

Min.,

2

1

875

29.96

58.46

0.13

0.42

0.22

0.14

0.42

0.15

MAPLE SIRUP. COXTROLS

Calculated to a moisture-free basis

u
o
.0

a
3

a

c.

a

cs
02

ALKALINITY

0

3

u

a

es

•a

>

0)

0)

fed

S3
CO

CO

c«

.3

CS

"O

a

3

CS

o>

53

0>

0

a

a>

o>

J3

0>

43

C3

1

0

~

XI

*^

.a

ej

0)

c

3
82

>

a

CO
O
EH

3
O

w

O

co
3

3

0

02

O

03

d

"3

es

G

%

%

%

%

%

%

%

6

95.41

0.97

0.94

0.41

0.53

36

94

0.92

1.76

3-24-09

20

95.37

1.58

1.25

0.38

0.87

45

129

1.56

0.24

4- 1-09

27

96.19

0.30

0.75

0.53

0.22

58

71

0.73

2.03

3- 7-10

41

97.08

1.03

0.77

0.37

0.40

59

85

0.8S

0.24

3-14-10

49

96.51

0.42

0.81

0.39

0.42

52

85

0.83

1.43

3-20 10

58

97.01

0.43

0.73

0.43

0.30

58

113

0.83

1.00

3-21-10

68

96.90

0.51

0.68

0.45

0.23

54

56

0.81

1.10

3-22-10

89

95.81

0.34

0.61

0.32

0.29

44

53

1.01

2.23

3-22-11

97

96.04

0.19

0.68

0.43

0.25

56

49

0.73

2.36

3-26-11

105

96.23

0.24

0.79

0.54

0.25

58

48

0.61

2.13

3-27-11

113

96.97

0.61

0.75

0.50

0.25

62

58

0.73

0.94

3-30-11

Av'ge,

96.32

0.60

0.77

0.41

0.36

51

77

0 86

1.45

Max.,

97 08

1.58

1.25

0.54

0.87

62

129

1.56

2.36

Min.,

95.37

0.19

0.61

0.32

0.22

36

48

0.61

0.24

Micro-organisms of Maple Sap 423

The average water content for the group is 34.06%. This
figure is about normal, yet individual sirups vary from 39.78 to
29.96%. The sucrose shows the usual variations depending on
the moisture content, ranging from 58.46 to 67.89, and averaging
63-5 1 %• When calculated to a dry matter basis these large
variations are eliminated and the average sucrose content becomes
96.32%. The invert sugar varies from 0.13 to 1.05. averaging
0.39%. Investigation has shown that there are but very small
amounts of invert sugar in the sap of the maple as it comes from
the tree, and, consequently, if quickly and properly concentrated,
flie resulting sirup should carry only a small amount. When
the invert sugar in maple sirup calculated to a moisture free
basis is less than 0.60%, it should not be attributed to excessive
micro-organic infection. No. 20 was obtained about the
middle of the season of 1909 from sap held 3 days in the cold ;
and its invert sugar percentage, 1.05, would indicate that the
sucrose had suffered a slight decomposition.

The minimum ash and malic acid value figures estab-
lished by the writer for pure Vermont maple sirup of standard
weight and density, together with similar data obtained by
Bryan for Vermont as well as for the United States-, arc shown
in the following table :

TABLE 17. MINIMUM ASH AND MALIC ACID VALUES FOB PURE MAPLE SIRUP

Calculated to a moisture-free basis

Jones (')

Bryan (■)

Bryan (2)

Vermont

Vermont

United States

samples

samples

samples

Total

ash,

0.77%

0.77%

O.G8%

Insoluble ash,

0.23%

0.23%

0.23%

Malic

acid

value,

0.61%

0.58%

0.31%

The agreement on the Vermont samples is very close. The
Jones standard was secured on samples made in 1904 and the
Bryan results on those obtained in 1909. Tn both cases they
are based on about 50 samples representing all grades of pure

lVt. Sta. Rpt. IS, p. 334 (1905).

aU. S. Dept. Agr., Bu. Chem., Bui. 134, pp. 74-75 (1010).

-t2-t Bulletin 167

sirup. The United States minimum is based on 395 samples,
representing all the important maple sugar producing sections.
Attention is called to the fact that the minimum for insoluble
ash for the entire United States is identical with the figure
twice obtained by different investigators on Vermont samples.

The ash and malic acid value data of the sirups in this and
subsequent groups will be discussed primarily with a view of
comparing the data with the standards just enumerated. It
should be remembered that many of the samples discussed
in the following groups are exceptional as to inoculation and
manufacture. Such divergencies from the normal as were
found will be noted and reasons assigned for the deficiencies.

The average total and insoluble ash contents of the group
on a moisture free basis are 0.77 and 0.36% respectively, with
variations for total ash from 0.61 to 1.25 and for insoluble ash
from 0.22 to 0.87%. "While the average of these 11 sirups is
normal as regards standards, yet in six cases the total ash is be-
low 0.77% (the Vermont standard) and in one case below the
United States minimum. The insoluble ash runs 0.01% low in
one instance, but in all others equals or exceeds the 0.23% figure.

The malic acid value meets the standard in every instance,
the average figure obtained being 0.86 ' \ , with extremes of 1.56
and O.61 '/< .

Attention is here called to the fact that, excepting Xos. 27.
58 and 68, the samples showing a low total ash had a relatively
small water content, and that in all cases the "niter"' had been
thoroughly removed by a full seven months' sedimentation. The
deficiencies occurring during 1910 and 191 1, if considered in con-
nection with color, flavor, and the remaining ash and malic acid
data, would not embarrass the analyst familiar with maple prod-
ucts.

The undetermined column shows variations from 0.24 to
2.36, averaging r.45%, which is about the usual amount found
in the ordinary run of good grade maple sirups.

Micro-organisms oe Maple Sap 1-25

GROUP 2. Incubator controls. The saps from which these
sirups were made were kept during the incubation period, gen-
erally not over 3 days, hefore being concentrated. They were
in many cases probably slightly contaminated from natural
sources and should, therefore, be considered only as of ordi-
nary quality. Sample 43 remained clear for 2 days, hut showed
a milk)- hacterial growth the third day hefore boiling. No. 65
likewise soured early and made a sirup of poor quality. All the
samples graded well as to color and in only two cases was the
flavor considered medium or poor.

-L2ii Bulletin 167

TABLE 18. MAPLE SIBUP. INCUBATOB CONTBOLS

u

0

<D

.3

a

sS

_.

c/:

>

3

fcc

(_

*O0

■3

—

c

4)

3

00

«

0

V

0

O

rt

O

3

a

V

0*

0Q

4-i

s

—

™

.

0

0

+J

O

tl

—

3

0

i

p*

0

a;

i-

O

r:

3

0

^

•a

2

"3

jS

S

"3

CJ

>
3

•**

0

0

00

c

Id

•'/.

^

GO

5

C-t

X

<i

—

%

%

^

Of

70

%

%

%

%

1

7

1

850

38.52

59.54

0.66

0.61

0.24

0.37

0.65

0.02

34

6

21

800

3G.06

G0.97

0.27

0.90

0.38

0.52

0.91

0.89

38

5

21

825

38.37

58.45

0.30

0.71

0.34

0.37

0.62

1.55

40

!t

2

750

34.89

G1.89

0.42

0.63

0.25

0.38

0.59

1.58

43

7

2

Mill

40.03

56.39

1.58

0.53

0.25

0.28

0.53

0.94

45

4

1

925

42.19

55.66

0.30

0.52

0.24

0.28

0.60

0.73

56

5

2

850

37.92

59.61

0.84

0.56

0.34

0.22

0.46

0.61

57

2

1

975

40.65

57.26

0.36

0.48

0.25

0.23

0.53

0.72

65

6

4

G25

42.03

53.27

2.04

0.60

0.25

0.35

0.63

1.43

9G

5

2

v-,11

27.S1

68.82

0.32

0.51

0.33

0.18

0.42

2.12

104

4

2

S75

29.46

68.42

0.29

0.49

0.33

0.16

0.41

0.93

112

:.

2

850

31.34

67.01

0.76

0.49

0.35

0.14

ti.31

0.09

120

5

O

775

38.97

58.06

0.35

0.44

0.28

0.16

0.44

1.74

Averag

e, 5.4

2.0

827

36.7S

60.41

0.65

0.57

0.29

0.28

0.55

1.04

Max.,

9

1

975

42.19

68.82

2.04

0.90

0.3S

0.52

0.91

2.12

Min.,

2

0
0

625

27.81

53.27

0.27

0.44

0.24

0.14

0.31

0.02

MAPLE SIBUP. I.NCUBATOK CONTROLS

Calculated to a moisture-free basis

C.

S

d

A

ALKALINITY

4)

d

rs

~

ID

J3

>

a>

d

£Z

a

00

a

be

d

—

0>

3

O0

00

d

a

d
0

3

c

3

O

_

£1

3

.q

3

0

w

—

O

d

3

O

3

0

t)

>

0

O

00

a

©

00

a

d

3
P

m

E-i

■f.

r.

►— 1

A

d
Q

%

%

%

%

%

%

%

1

96.84

1.07

0.99

0.39

0.60

55

120

1.06

0.04

3-24-09

34

95.36

0.42

1.41

0.59

0.82

69

153

1.42

1.39

3- 7-10

38

94.80

0.48

1.15

0.55

0.60

74

119

1.00

2.57

U

40

95.05

0.64

0.96

0.38

0.58

62

107

0.91

2.44

M

43

94.19

2.64

0.8S

0.41

0.47

58

88

0.88

1.41

3-14-1"

45

96.28

0.51

0.89

0.41

0.48

60

96

1.04

1.2S

a

56

96.02

1.35

0.90

0.54

0.36

58

95

0.73

1.00

3-20-10

57

96.47

0.60

0.81

0.52

0.39

61

81

0.90

1.22

C4

65

91.89

3.52

1.03

0.43

0.60

60

122

1.09

2.47

3-21-10

96

95.33

0.44

0.71

0.46

0.25

55

49

0.65

2.87

3-22-11

104

97.00

0.41

0.69

0.47

0.22

62

42

0.60

1.30

3-26-11

112

97.60

1.11

0.71

0.50

0.21

59

47

0.44

0.14

3-27-11

120

95.12

0.57

0.71

0.45

0.26

66

75

0.72

L'>s

3-30-11

Average

, 95.55

1.04

0.90

0.46

0.44

61

92

0.87

1.64

Max.,

97.60

3.52

1.41

0.59

0.S2

74

153

1.42

2.87

Min.,

91.89

0.41

0.69

0.38

0.21

55

42

0.44

0.04

Micro-organisms of Maple Sap t27

The moisture content in these thirteen samples varies from
27.81 to 42.19 with an average of 36.78%.

The sucrose ranges from 53.27 to 68.82 and averages
60.41%. No. 65, which contains the lowest sucrose figure, is
in consequence high in both water and invert sugar.

The invert sugar averages 0.65% with a miximum of 2.04
and a minimum of 0.27%. None of the samples, save Nos. 43
and 65, show marked evidences of souring previous to boiling.
No. 1 was purposely evaporated at a slow rate, about 6 hours,
or four times the usual interval, being taken. Its low invert
sugar content, 0.66%, together with its satisfactory flavor and
color, indicate that the time taken in boiling this fairly purr
sap produced no marked inversion effect on the sucrose.

Four of these samples, Nos. 96, 104, 112 and 120, when
calculated to a moisture free basis, show a deficiency of total
ash, two of which, Nos. 104 and 112, are just under the limit
for insoluble ash, while one, Xo. 112, is low in malic acid value.
In three of these four cases the concentration of the sirup was
carried too far, the moisture content ranging from 27.81 to
31.34%. The malic acid value figure, 0.44%, is the lowest ever
observed by the writer on pure Vermont goods.

Group 3. Inoculated with non-fluorescent bacteria. These
organisms are common in nature. When they occur in the sap
and are allowed to develop, they injure the flavor more than they
do the color of the resulting sirup. Two of the four samples
were rated medium as to flavor, and two, good. The color of all
four was exceptionally light, easily being classed No. 1 as com-
mercially graded.

42S

Bulletin 167

TABLE 19. MAPLE SIRUP. INOCULATED WITH NON-FLUOBESCENT BACTEKIA

0>

a>

,0

—

r.

f—

a

£■.

>

O

eS

.a

a

rt

a

<D

so

a

03

OS

SJ

"3

a

lA

o>

.Q

C3

7"

00

.

0

—

0

»— t

pC

O

a

0

0

0

0
>

r.

O

a

o3

a
P

%

%

%

%

%

%

'>

%

9

5

2

850

33.26

G3.81

0.52

0.57

0.26

0.31

0.47

1.37

Hi

G

750

32.21

G4.98

0.52

0.78

0.27

0.51

0.60

0.91

12

G

•>
O

750

3G.35

G0.92

1.42

0.76

0.27

0.49

0.73

—.18

55

5

2

850

36.97

59.73

1.50

0.63

0.26

0.37

0.48

0.69

Ave rag

e, 5.5

2.5

800

34.70

62.36

0.99

0.69

0.27

0.42

0.57

0.69

Max.,

6

q
0

850

36.97

64.98

1.50

0.78

0.27

0.51

0.73

1.37

Min.,

5

2

750

32.21

59.73

0.52

0.57

0.26

0.31

0.47

—.IS

MAPLE SIRUP. INOCULATED WITH KTON-FLUOBESCENT BACTERIA

Calculated to a moisture-free basis

ALKALI X ITT

<u

a

a

,

J3

a

w

A

>

a>

a

a

fcJO

a

a

0)

fit

ei

01

o

a

a

<a

<x>

0}

.0

tt)

X3

a

0

_ 1

A

S

A

s

0

+j

a

5

a

5Q

a

Q

O

O
m

O
K3

c

a

1— 1

"3

*5

a
P .

G

%

%

^O

%

%

%

%

9

95.61

0.78

0.85

0.39

0.46

51

117

0.70

2.06

3-27 09

10

95.86

0.77

1.15

0.40

0.75

35

145

0 88

1.34

•■

12

95.71

2.23

1.19

0.42

0.77

41

145

1.14

—.27

••

55

94.76

2.3S

1.00

0.40

0.60

57

123

0.75

1.11

3-20-11

Averag

e, 95.50

1.52

1.06

0.42

0.64

46

132

0.87

1.05

Max.,

95.86

2.38

1.19

0.42

0.77

57

145

1.14

2.06

Miu.,

94. 7G

0.77

0.85

0.39

0.46

35

117

0.70

—.27

Micro-organisms of Maple Sap t29

The moisture content of these samples was quite uniform,
a variation of less than 5', being found. Its average was

3470%-

The sucrose varied from 59.73 to 64.98, averaging 62.36^ ■

The invert sugar, while averaging higher than in the pre-
viously mentioned controls, did not show such extremes. The
average of 0.99% as analyzed, equivalent to 1.52% on a dry
hasis, is clearly indicative of the fact that the organisms used
were not active inverters of sucrose. The records show that the
sap from which sirup 55 was made had a bacterial count of nearly
3.000,000. While apparently inactive as regards inversion they
did, however, affect the flavor of the product, causing it to fall
off 1.5 points from the control.

The ash and malic acid value data in all cases exceed the
usual standard. It should he noted, however, that the first three
samples, Nos. 9, 10 and 12, were from the 1909 crop, and, as
has been mentioned, no particular care was taken to remove all
niter as was the case with No. 55.

Group 4. Inoculated with pink cocci. The presence of
these organisms in maple sap is decidedly detrimental to the pro-
duction of a first class sirup. They seriousl) affect the flavor
hut have no marked influence on the color.

430

Bulletin 167

TABLE 20. MAPLE SIRUP. INOCULATED WITH PINK COCCI

u

0)

0

.a

n

cd

*w

a

■^

ta

>

o>

3

a

4)

03

CO
03

cd
0

■a

0

c

a

v

0

4>

2

□3

0

*j

2

3

-

a

03
SO

0
0
O

fa

O
DQ

0

s

H2

>
a

cd

0
Eh

3

0

CO

0

CO

0

1— (

1

■a

a
P

%

%

%

%

%

%

%

%

4G

6

2

825

38.42

58.55

0.65

0.63

0.27

0.36

0.60

1.15

47

6

O

750

40.64

52.72

4.69

0.60

0.27

0.33

0.52

0.83

50

0

21

800

40.03

56.17

1.32

0.50

0.23

0.27

0.50

1.48

51

4

2

875

37.65

60.67

0.41

0.51

0.31

0.20

0.49

0.27

107

b

4

GOO

27.90

68.15

0.50

0.51

0.36

0.15

0.42

2.52

54

7

22

750

38.30

58.07

1.67

0.51

0.27

0.24

0.44

1.01

Avcrag

0, 5.7

2.G

775

37.15

59.05

1.54

0.54

0.28

0.26

0.50

1.22

Max.,

7

4

875

40.64

68.15

4.69

0.63

0.36

0.36

0.60

2.52

Min.,

4

2

600

27.90

52.72

0.41

0.50

0.23

0.15

0.42

0.27

MAPLE SIRUP. INOCULATED WITH PINK COCCI

Calculated to a moisture-free basis

u

0)
CO

p

V

3
W

cd
CO

>
a

.3

CO

ed

"3

0
H

a

CO

cd

CO

3

3

O
03

S3

CO

cd

0)

—
3

"o

CO

3
1— t

ALKALINITl

CD

3

>

03

3

a

1

0
o>

-a
a
P

a
3

a

a

cd

S3

CO

ed

0)

3

3

"B
xn

a

cd

3
3
0

CO

a

CD

cd
P

46

%

95.07

1.05

%
1.02

0.44

%

0.58

162

113

0.97

%
1.89

3-14-10

47

88.81

7.90

1.01

0.46

0.55

64

107

0.88

1.40

f(

50

93.83

2.20

0.83

0.38

0.45

62

95

0.S3

2.31

3-20-10

51

97.30

0.65

0.82

0.50

0.32

63

73

0.79

0.44

••

107

94.52

0.69

0.71

0.50

0.21

57

34

0.58

3.50

3-27-11

54

94.11

2.71

0.82

0.43

0.39

56

75

0.71

1.65

3-20-10

Averag

e, 93.94

2.45

0.86

0.45

0.41

61

83

0.79

3.96

Max.,

97.30

7.90

1.02

0.50

0.58

G4

113

0.97

3.50

Min.,

88.81

0.G5

0.71

0.38

0.21

56

34

0.5S

0.44

Micro-organisms of Maple Sap 431

The moisture content of the six sirups averaged 37.15 with
extremes of 40.64 to 27.90% . The minimum amount is found in
No. 107, the only 191 1 sample in this series.

The sucrose averages 59.05 and the extremes are 52.72
and 68.15%. The sample, No. 47, with the highest moisture con-
tent carries the lowest sucrose percentage. The relatively
large amount of invert sugar present is also a contributing fac-
tor.

The invert sugar shows a surprising variation of from
0.41 to 4.69 with an average of 1.54%. This maximum figure,
in No. 47, when calculated to the moisture free basis is equivalent
to 7.90''. This result may be attributed to the action on tin-
sucrose of a certain strain of pink coccus. Other strains did not
give as positive evidences of sucrose inversion.

No. 107, as compared with standard, is slightly deficient
in total and insoluble ash and in malic acid value.

Group 5. Failures. The saps from which the sirups thus
characterized were made, were variously inoculated, but the intro-
duced organisms could not be recovered at the close of the in-
cubation period. The natural infection may have suppressed their
development or the inoculating organisms may have been killed
by those normally present. Whatever the infection, it had no
marked influence upon the results in the invert sugar column.

The color averaged a little poorer in these samples than
in those previously mentioned under the head "incubator control,"
while flavor showed a slight gain.

/

432

Bulletin 167

TABLE 21. MAPLE SIRUP. FAILUKES

MAPLE SIRUP. FAILURES

Calculated to a moisture-free basis

0)

D

3

£1

.3

rt

-=

g

--

cs

<*^

c;

a

O

M

X

■s.

a

a

-C
w

g

'-*

0>

K

O)

£>

CS

B

0

OJ

+->

_-

3

3

O

4-1

a

cS

0
c

8j

0
'■J

*o

3

0>

>

CS

0

O
v.

-

c

m

0

fe

•Jl

f<,

•7.

>-<

H

v.

~

r«5

—

%

%

'<

%

%

%

%

%

31

G

2

825

38.21

59.70

0.19

0.70

0.34

0.36

0.34

0.8G

32

G

2

825

37.37

60.07

0.29

0.71

0.25

0.46

0.65

(1.91

• >•»

» > a

7

2

800

35.96

61.23

0.15

0.7G

0.34

0.42

0.64

1.2G

36

9

21

725

37.52

(111. OS

0.38

0.65

0.27

0.38

0.45

0.92

37

11

21

G75

34.43

62.87

0.41

0.73

0.24

0.49

0.65

0.91

39

7

2

800

36.34

61.23

0.17

0.71

0.22

0.49

0.62

0.93

1 OG

7

• >

725

30.48

67.G4

0.46

0.48

0.33

0.15

0.41

0.53

108

4

O

800

29.64

G7.10

0.63

0.46

0.32

0.14

0.35

1.82

Averag<

', 7.ii

2.3

772

34.99

62.48

0.::::

0.65

0.29

0.36

0.52

1.03

Max.,

11

*>
0

825

38.21

67.64

0.63

0.76

0.34

0.49

0.65

1.82

Min.,

G

2

675

29.64

59.70

0.15

0.46

0.22

0.14

0.34

11.:,:;

ALKALINITY

Cv

£>

a
3

a

a

cs
w

0)
O

02

cs
s

y.

03

7s

0

CS

■J.

a

QQ

CS

3

.3

03

C3
0)
3
3

O
f.

a

W

CS

3

~z
m

>

CS

73

M

a

a

01
4)

a

CO

-
Q

31

9?
96.62

0.30

<<

1.14

c1

70

0.55

'7c
0.59

62

1 Q9

1 ->. >

0.55

%
1.39

;;- 7-1 «

32

95.91

0.4G

1.13

0.39

(1.74

54

102

1.(13

1.47

• »«»

95.61

0.23

1.18

0.52

0.66

62

151

0.99

1.99

36

96.16

0.G1

1.06

0.44

0.62

57

105

0.7::

1.44

37

:t5.8S

(1.04

1.11

0.36

0.75

51

79

0.99

1.38

39

96.18

0.2G

1.12

0.35

0.77

07

120

0.97

1.47

106

97.30

0.66

0.69

0.47

0.22

58

56

0.59

0.76

3-27-11

108

95.37

0.90

0.65

0.45

0.20

58

37

0.49

2.59

a

Ave rag

e, 96.13

0.51

1.00

0.45

0.55

59

98

0.80

1.50

Max.,

97.30

0.90

L.18

0.55

0.77

07

151

1.03

2.59

Min.,

95.37

0.23

0.65

0.35

0.20

51

37

0.49

0.76

M icro-org w tSMS OF M \ im.i-; Sap t33

The moisture in the sirups of this group varies from 29.64
to 38.21, and averages 34.99%.

The sucrose averages 62.48^ . equivalent, on a moisture tree-
basis, to 96.13' i .

The invert sugar percentages were low, the average on a
moisture free basis being 0.51%, the highest, 0.90, the lowest
0.23'/. This checks closely with the results shown in tables [6
and 18, with which the data secured with the samples of this
group are comparable.

The total and insoluble ash contents are low of standard in
two of the eight samples and the malic acid value is deficient in
three samples.

Group 6. Inoculated ivith red yeasts. These organisms are
quite common, particularly toward the close of the season. The
evaporating sap and sirup containing them give off an offensive
yeasty odor. They seriously impair the flavor and in many
instances cause the color to grade several shades darker than
it otherwise would ; and they also materially increase the invert
sugfar content because of their action on the sucrose.

•±34 Bulletin 167

TABLE 22. MAPLE SIRUP. INOCULATED WITH RED YEASTS

u
o>

a

a

0>

S
to

co

to

.3

GO

—

0)

3

"5
>

£
a

S

0>

ti

~

4)
09

-t->

a

OJ

3

Ed

01

3.

E-i

0

CD

-t-^

O

i-i

^^

3

__

V

0

a

O

3

>

0

0

"0

a

>
a

a

0

3

O

CO

a

"5

•—

a

oa

0

E

VI

re,

vi

t-H

EH

X

*5

P

%

%

%

%

%

%

%

%

14

10

2

725

33.17

63.69

2.01

0.87

0.31

0.56

0.59

-0.33

62

9

4

550

40.18

53.40

4.37

0.51

0.22

0.29

0.49

1.05

15

8

2

750

38.88

57.00

1.82

0.77

0.26

0.51

0.92

0.G1

114

5

3

775

30.69

6G.75

0.70

0.49

0.34

0.15

0.39

0.98

IS

10

2

725

30.17

G4.00

2.18

0.72

0.27

0.45

0.72

2.21

35

Vi

21

650

32.6G

G4.06

0.77

0.64

0.25

0.39

0.56

1.31

»i'

5

2-

800

41.31

54.74

1.26

0.69

0.36

0.33

0.72

1.2S

59

7

■>

725

38.97

57.82

1.24

0.59

0.30

0.29

0.54

0.84

GO

G

750

38.1G

59.02

1.09

0.52

0.33

0.19

0.67

0.54

61

9

'i
O

G75

35.92

61.39

1.13

0.54

0.30

0.24

0.47

0.55

99

7

4

600

28.11

G8.17

1.47

0.54

0.32

0.22

0.45

1.26

G3

8

•»

700

37.23

55.90

4.79

0.55

0.27

0.28

0.50

1.03

G4

8

700

35.88

58.76

3.76

0.51

0.27

0.24

0.46

0.63

Average

i, 8

2.8

703

35.48

60.36

2.05

0.62

0.30

0.32

0.61

0.88

Max.,

12

4

800

41.31

68.17

4.79

0.87

0.36

0.56

0.92

2.21

Min.,

5

2

550

28.11

53.40

0.70

0.49

0.22

0.15

0.39 -

-0.33

MAPLE SIRUP. INOCULATED WITH RED VI AM

Calculated to a moisture-free basis

v.
O

3

02

M

to

<D
>

a

.a
m

a

"3

0

H

ti

CO

ej

0>

3
0

.a

CO

03

01

3

CO

ALKALINITY

3

"5

>

•a

CJ

Bj

O

"3

OJ

a
1

OJ

+j

.2

ed

a

3

a

CD

a

vi

CO

03
o>

3

3-
O
X

■3

CO

ea

£
3
3

3

to

a

14

95.30

%

3.01

1.30

%
0.46

%

0.84

33

144

%

0.88 -

%
—.49

4- 1-09

62

89.27

7.30

0.86

0.37

0.49

51

103

0.83

1.74

3-21-10

15

93.26

2.98

1.26

0.43

0.83

69

164

1.50

1.00

4- 1-09

114

96.31

1.01

0.71

0.49

0.22

61

52

0.56

1.41

3-30-11

18

91.65

3.12

1.03

0.39

0.64

49

112

1.03

3.17

4- 1-09

35

95.13

1.15

0.95

0.37

0.58

67

114

0.84

1.93

3- 7-10

42

93.27

2.14

1.18

0.61

0.57

58

122

1.23

2.18

3-14-10

59

94.74

2.03

0.97

0.49

0.48

65

99

0.88

1.38

3-21-10

60

95.44

1.76

0.85

0.53

3.32

58

91

1.08

0.S7

u

61

95.80

1.77

0.84

0.46

0.38

64

81

0.73

0.86

II

99

94.82

2.05

0.75

0.4 I

0.31

57

63

0.67

1.71

5-26-11

63

89.06

7.63

0.88

0.43

0.45

63

91

0.80

1.63

3-21-10

64

91.64

5.86

0.79

0.42

0.37

54

82

0.72

0.99

C<

Avcrag<

•, 93.54

3.18

0.96

0.47

0.49

58

101

0.91

1.41

Max.,

96.31

7.63

1.30

0.61

0.84

69

164

1.50

3.17

Min.,

89.06

1.01

0.71

0.37

1.22

33

52

0.56

-.49

Micro-orc.anisms of Maple Sap 435

The moisture shows the usual variations, averaging- 35.48%.

The sucrose averages 60.36, with a maximum of 68.17
and a minimum of 53.40%.

The invert sugar averages 2.05 and extremes range from
0.70 to 4.79%. This maximum is equivalent to 7.63% on a mois-
ture free basis. The count secured on the plated sap previous
to boiling seems to bear no direct relation to the amount of in-
vert sugar. The count includes both bacterial and yeast colonies,
however, so that the figures do not express the relative infection
with yeasts. The variations in the invert sugar content may be
assigned partly to differences in the individual strains employed
and partly to the different degrees of infection secured. (See
table 9, pages 392 to 395, Nos. 42, 63 and 64).

The total ash in Nos. 114 and 99 is below standard and the
insoluble ash in No. 114 is also 0.01% low. The malic acid
value is likewise a few points shy in No. 114. Both these samples
were quite concentrated, their water contents being 30.69 and
28.11% respectively.

Group 7. Inoculated with gray yeasts. These organisms
are closely associated with the red yeasts previously mentioned
and, like them, are quite commonly found late in the sugar sea-
son. Their action is detrimental to a good flavored sirup, four
out of the eight samples being graded 4 in flavor, correspond-
ing to poor. In two cases the color was considerably darkened,
being classed 10 and 11, corresponding to a No. 2 commercial
grade. The invert sugar was increased in every case and a
maximum percentage of 19.15% was found in No. too.

436

Bulletin 167

TABLE 23. MAPLE SIRUP. INOCULATED WITH GRAY YEA8TS

.0

s

2

0

Si

r:

m

>

c

^

at

cl

_

GQ

H3

0>

a

Si

s>

"Z

—

01

c

.Q

s

3

•

0

+J

c

i*

1 —

.fl

0

0

—

i.

OS

£

O

t-j

0

cj

0
0

0

>

O

ri

CD

S

od

— '

&

SO

rs

■7.

*- 1

H

■J.

i»5

%

%

%

%

%

%

%

%

13

7

2

800

36.05

59.69

1.62

0.79

0.30

0.49

0.92

0.93

28 '

11

4

500

35.93

53.07

7.79

0.79

0.28

0.51

0.67

1.75

109

10

4

525

32.44

45.49

19.15

0.60

0.33

0.27

0.61

1.71

48

0

2=

775

38.49

r> 7 . 7 .">

1.44

0.63

0.35

0.28

0.51

1.18

52

5

2

850

38.37

59.68

0.75

0.56

0.30

0.26

0.51

0.13

53

G

750

41.50

53.90

2.32

0.49

0.32

0.17

0.60

1.19

110

6

4

G25

30.94

67.05

1.20

0.46

0.30

0.16

0.46

—.11

115

7

4

GOO

31.04

65.61

1.05

0.50

0.35

0.15

0.43

1.37

Average

•, 7.3

3.2

G78

35.60

57.78

4.42

0.60

0.32

0.28

0.59

1.01

Max.,

11

4

850

41.50

67.05

19.15

0.79

0.35

0.51

0.92

1.75

Min.,

5

2

500

30.94

45.49

0.75

0.46

0.28

0.15

0.43

—.11

MAPLE SIRUP. INOCULATED WITH GRAY YEAST

Calculated to a moisture-free basis

.0

a

a

ALKALINITY

U

r.

J3

>

:.

C2

a

v.

b£>

■/.

A

<r.

~

4)

p

95

■J.

-

01

4;

i
2

—

£

O

-tJ

„

3

2

~

u

—

0

a)

^

s

p

*

>

=

c

0

A

O

J.

a

G

M

a

-/.

H

CO

h—

X

hH

(*i

H-

1

-;

%

%

%

%

%

%

13

93.34

2.53

1.24

0.47

0.77

56

141

1.44

1.45

4-10-09

28

.82.83

12.15

1.23

0.44

0.79

62

151

1.04

2.75

3- 7-10

109

67.33

28.35

0.88

0.49

0.39

50

102

0.90

2.54

3-27-11

48

93.89

2.35

1.03

II.5S

0.45

60

102

0.83

1.90

3-14-10

52

96.84

1.21

0.90

0.49

0.41

63

85

0.82

0.27

3-20-10

53

92.13

3.96

0.84

0.54

0.30

62

71

1.03

2.04

••

110

97.09

1.74

0.67

0.44

0.23

58

52

0.65

--.15

3-27-11

115

95.14

1.52

0.72

0.51

0.21

61

53

0.62

2.00

::-::<i-n

A. veras

e, sii. 72

6.86

0.94

0.50

ii.ll

59

95

0.92

1.56

Max.,

97.09

28.35

1.24

0.58

0.79

63

i:.i

UH

2.75

Miu.,

67.33

1.21

0.67

ill!

0.21

50

52

0.62

—.15

Micro-organisms of Maple Sap *;>T

The moisture content varied from 30.94 to 41.50 and aver

aged 35-6o%.

The sucrose averaged 57.78% with extremes of 45.49 and
67.05%. The low figure which was obtained on No. 109 is due
mainly to the high invert sugar content of [9.15%, equivaleni
on the moisture free material to 28.35$ ■ This was the largest
invert sugar percentage obtained with any of the samples ex-
amined. Reference to the field notes shows that the inoculating
organism multiplied rapidly and was an acid producer, a con-
dition which doubtless served to hasten the sucrose inversion.

The total ash on the moisture free basis shows a deficiency
in two cases, Nos. no and 115. The insoluble ash is also low in
No. 115. The malic acid value meets the standard in every case.

Group 8. Inoculated with fluorescent bacteria. These or-
ganisms occur more commonly in maple sap than do any of the
others used in this work. They grow well at low temperatures and
hence should be guarded against early in the season. They im-
pair the flavor somewhat although not as seriously as several
other groups. Nine of the 22 samples reported were graded
as No. 2 in flavor, indicative of a good sirup, six as No. 3, a good
medium sirup, while seven were classed as No. 4, corresponding
to poor. The color was seriouly injured by this fluorescent
group. They cause but little if any inversion of the sucrose
and never produce the objectionable yeasty taste so characteris-
tic of many contaminated sirups.

438

Bulletin 167

TABLE 24.

MAPLE SIRUP. INOCULATED WITH FLUORESCENT BACTERIA

CD
.Q

a

a
0.

a

V2

0
"o

0

0

>

s

O

O

w

05

1

CO

0

s

w

be
oo

>
s

"Hi

CS

;-

00

as

s

5C

A

ID

as

3
00

a

0

"3

>

V

a
"3

a

•a

a

1

HI

<u

•a
a
P

%

%

%

%

%

' <

%

%

2 '

14

4

425

31.01

66.77

1.07

0.71

0.30

0.41

0.54

—.10

102

I

4

GOO

28.81

68.11

0.81

0.58

0.41

0.17

0.36

1.31

0
0

11

2

700

31.15

66.60

1.44

0.81

0.32

0.49

0.59

—.59

103

7

4

GOO

28.75

67.97

0.96

0.52

0.32

0.20

0.43

1.37

4

9

2

750

30.69

66.91

1.08

0.71

0.33

0.38

0.65

—.04

5

11

2

700

33.55

64.56

0.96

0.59

0.27

0.32

0.44

—.10

7

10

2

725

32.72

G4.G9

1.10

0.64

0.20

0.44

0.59

0.26

8

10

2

725

33.9G

64.15

0.76

0.58

0.29

0.29

0.50

0.05

101

8

3

700

28.36

69.10

0.67

0.51

0.32

0.19

0.38

0.98

29

8

2*

750

37.31

59.50

0.24

0.67

0.24

0.43

0.54

1.74

30

S

2

775

32.91

64.90

0.60

0.71

0.30

0.41

0.63

0.25

44

9

3

G75

38.72

57.01

0.86

0.64

0.25

0.39

0.70

2.07

100

7

3

725

27.7G

69.59

0.67

0.57

0.39

0.18

0.40

1.01

G9

9

3

G75

37.17

58.91

1.00

0.76

0.24

0.52

0.79

1.37

70

11

4

500

36.72

59.15

1.56

0.80

0.34

0.46

0.70

1.07

71

14

4

425

37.87

58.20

0.36

*_

*f

*_

0.66

• ■ ■ ■

72

13

4

450

36.57

60.30

0.28

0.66

0.19

0.47

0.66

1.53

73

G

2

825

33.31

65.00

0.45

0.55

0.32

0.23

0.48

0.21

91

G

3

750

2G.76

69.40

0.29

0.53

0.32

0.21

0.66

2.36

92

7

3

725

28.4G

67.96

0.37

0.58

0.37

0.21

0.53

2.10

93

G

2

825

28.06

68.65

0.31

0.60

0.41

0.19

0.39

1.99

94

7

4

GOO

28.51

67.69

0.37

0.51

0.30

0.21

0.46

2.46

Average

\ 9

2.9

G65

32.23

64.79

0.74

0.63

0.31

0.32

0.55

1.06

Max.,

14

4

825

38.72

69.59

1.56

0.81

0.41

0.52

0.79

2.46

Min.,

6

2

425

2G.76

57.01

0.24

0.51

0.19

0.17

0.36

—.59

Micro-organisms of Maple Sap

439

MAPLE SIBUP, INOCULATED WITH FLUORESCE!*! BA( L'EBIA

Calculated to a moisture-free basis

u

u

S3
w

ALKALINITY

4>

3

"5

>

-a

IK

Si

a

S3

3

aS

si

cd

J3

09

■3

a

s

3
03

S3

09

OS

0)

DQ

4)

'S

i

1>

0>

rt

(U

2

a;

S:

cd

^

"c.

09

o

+->

_

3

3

3

3

V

4>

a

o

0)

cd

o

3

O
m

3

3
o

O
CO

c

i

■a

3

01

cd

w

03

I— <

trl

w

>— i

Ul

>— i

<-.

P

Q

%

%

%

%

%

%

%

2

96.79

1.55

1.03

0.44

0.59

49

113

0.78

-0.15

3-24-09

102

95.67

1.14

0.S2

0.58

0.24

64

58

0.50

1.87

3-26-11

•i

96.73

2.09

1.18

0.47

0.71

52

113

ii. m;

-0.86

3-24-09

103

95.38

1.35

0.73

0.45

0.28

56

65

0.60

1.94

3-26-1 1

4

96.54

1.56

1.02

0.48

o.r>4

52

110

0.93

0.05

3-24-09

5

97.16

1.44

0.89

0.41

0.48

48

99

0.67

-0.16

3 24 09

7

96.16

1.64

0.95

0.30

0.65

54

104

0.88

0.37

3-27-09

8

97.14

1.15

0.88

0.44

0.44

42

82

0.75

0.08

(1

101

96.45

0.94

0.71

0.44

0.27

60

56

0.53

1.37

3-26-11

29

94.91

0.38

1.07

0.39

0.68

61

131

0.87

2.77

3- 7-10

30

93.75

0.89

1.06

0.45

0.61

63

122

0.93

3.37

ft

44

93.03

1.40

1.06

0.41

0.65

61

122

1.15

3.36

3-14-10

100

96.21

0.93

0.79

0.54

0.25

61

50

0.55

1.52

3-26-11

69

93.87

1.59

1.21

0.38

0.83

69

153

1.26

2.07

3-22-10

70

93.47

2.46

1.26

0.53

0.73

62

176

1.10

1.71

ft

71

93.67

0.58

*

*

*

*.

* .

1.06

It

72

95.07

0.44

1.03

0.29

0.74

64

150

1.03

2.43

(1

73

97.47

0.68

0.83

0.48

0.35

70

66

0.71

0.31

tt

91

94.76

0.40

0.72

0.44

0.28

58

58

0.90

3.22

3-22-11

92

95.00

0.52

0.81

0.51

0.30

57

76

0.74

2.93

«(

93

95.43

0.43

0.83

0.56

0.27

70

54

0.54

2.77

ft

94

94.68

0.52

0.72

0.41

0.31

57

45

0.64

3.44

(I

Averag

e, 95.42

1.09

0.93

0.46

0.47

59

95

0.82

1.74

Max.,

97.47

2.46

1.26

0.58

0.83

70

176

1.26

3.44

Min.,

93.03

0.38

0.71

0.29

0.24

48

45

0.50

—.86

*Not determined through inadvertence.

440 Bulletin 167

The moisture ranged from 26.75 to 3&-72< averaging 32.23$ .

The sucrose variations, inasmuch as the invert sugar is low,
follow the water content quite closely. The minimum found was
57.01, the maximum 69.59 an(' tne average 64.79' < .

The invert sugar variations of from 0.24 to 1.56, witli the
average of 0.74%, indicate quite conclusively that the inoculating
organisms were not invert sugar formers.

The total ash as recorded in the moisture free basis table
is below standard in Nos. 103, 10 r, 91 and 94. The insoluble
ash meets all requirements in every case, while the malic acid
value is low in four instances, Nos. 102, 101, 100 and 93.

Group 9. Composites. The saps from which this group
of sirups was made were severally inoculated with from two to
six organisms, hence the results are the product of a mixed infec-
tion. The object held in view in this procedure was to ascertain
whether the presence in considerable numbers of two or more
organisms would serve to stimulate or to reduce specific indi-
vidual action. The flavor of all four samples was impaired,
but the color was not as seriously affected. It is interesting to
note that No. 19, inoculated with a mixture of fluorescent bac-
teria, yeasts and molds, contained a high invert sugar percentage
and that the gray yeast used for inoculation purposes was the
same organism employed in the inoculation of Nos. 13, 28 and
i<>0 (table 23). in which the highest invert sugar contents ob-
tained in this investigation were found.

Micro-organisms <>k Mai'i.h Sap

HI

TABLE !'">. MAPLE SIBUP. COMPOSITES

-

O)

■-
.0

A

7;

—

s

-~

•/.

>

<3J

s

tJC

a

T

=

0)

9

0>
•J.

0Q

a>

4)

3

-

~

—

O

a)

+-»

~—

£l

— *

i)

od

o

cd

©

*o

O

<1>
>

o

"o

a

«

—

a

/.

O

, fa

02

f^

B2

HH

H

02

^.

—

%

%

%

%

%

%

%

%

19

9

G75

.32. 4G

G3.00

2.7G

0.G9

0.23

0.46

0.86

0.23

116

8

4

575

29.S9

G7.04

0.4S

0.60

(1.4.-.

0.15

0.46

i.r.::

118

7

4

000

29.30

G7.G6

0.G9

0.59

0.41

II. is

0.41

1.35

119

7

2

800

28.75

68.59

0.58

0.4G

0.30

0.16

0.42

L.20

Average

', 7.7

3

GG3

30.10

GG.57

1.13

0.59

0.35

0.24

0.53

L.08

Max.,

9

4

800

32. 4G

G8.59

2.7G

0.69

0.45

0.46

0.86

1.53

Min.,

7

2

575

28.75

G3.00

0.48

0.46

0.23

0.15

0.41

0.23

MAPLE SIBUP. COMPOSITES

Calculated to a moisture-free basis

19
in;
lis
119

%
93.28
95.62
95.69
96.27

Average, 95.22
Max., 96.27
Min., 93.28

%
4.09
0.68
0.98
0.81

1.64

4.09
0.68

%
1.02
0.86
0.84
0.64

%
0.34
0.65
0.59
0.42

%
0.68
0.21
0.25
0.22

0.S4 0.50 0.34
1.02 0.65 0.68
0.64 0.34 0.21

50
54
64
59

57

64

.10

127
54
64
48

73

127
48

%
L.27

0.65
0.58
0.59

%
0.34
2.19
1.91
1.69

0.77 1.:.::
1.27 2.19
0.58 0.34

4- L-09
3-30-1 1

442 Bulletin 167

The moisture content of the four samples in this group is
low, indicating a greater density than is called for in standard
sirup. It averaged 30.10% with extremes varying less than

4%-

The average sucrose content was 66.57%.

The invert sugar ranges well within the limits for average
sirup, the high figure of 2.76% in No. 19, as has hitherto been
pointed out, being occasioned by the activity of a gray yeast
which in this and other instances exerted a pronounced invert-
ing action on the sucrose.

The total ash is below standard in No. 119, the insoluble
ash is low in Nos. 116 and 119, and the malic acid value just
under the limit in Nos. 118 and 119.

Group 10. Inoculated with Bacillus accris (new specie^ 1.
This organism causes a distinct type of stringiness in maple sap.
Tt produces acid, inverts sucrose and forms gas. Fortunately
for the sugar maker it does not appear to be very common in
the sugar orchards. It exerts a most injurious effect on the
flavor, two of the three samples inoculated scoring 4 and the
other, 3. The color, while showing a depreciation from the con-
trol, does not appear to be affected to any great extent, for all
three samples would have been commercially classed No. 1 in
this particular. All the sirups had a cloudy appearance and the
precipitated niter and other impurities settled out with difficulty.
Previous to boiling the sap was quite stringy, but the resulting
sirups were not especially viscid.

Micro-organisms o£ Maple Sap

443

TABLE 26. MAPLE SERUP. INOCULATED WITH Bacillus (ICCris

MAPLE SIRUP. INOCULATED WITH BdvillUS (ICdis

Calculated to a moisture-free basis

u

CD

X>

a

u

xi

0)

3

■8

3

■a

od

3

a

d)

bo
3

xi

CO

O)

a

4)

tj

<L>

eS

o>

XI

od

4-»

a

h

o

41

+J

O

b

~~

A

o

a

©

*©
u

>

O

o
K

O
3
02

>

a
i— i

od

o

3
O

w

O
ED

e

1— 1

■a

a
P

%

%

%

%

%

%

%

%

11

7

4

600

32.56

64.82

1.16

0.57

0.29

0.28

0.48

0.41

95

6

3

750

27.91

65.49

3.63

0.66

0.34

0.32

0.51

1.80

90

7

4

600

29.31

61.30

5.09

0.87

0.37

0.50

0.75

2.68

Average

i, G.7

3.7

650

29.93

63.87

3.29

0.70

0.33

0.37

0.58

1.63

Max.,

7

4

750

32.56

65.49

5.09

0.87

0.37

0.50

0.75

2.68

Min.,

6

3

600

27.91

61.30

1.16

0.57

0.29

0.28

0.48

0.41

b

u

xi

ALKALINITY

O)
3

3

>

■s

Xi

a

XI

3

be

.3

od

X!

ed

•a

a

3

to

a

a>

od

4)

•*H

a

01

<D

cd

4>

X

a>

XI

Ed

Ll

C

o

b

_

XI

*^

Xl

3

o

4-1

a

Ed

V

09

>

a

od

^-<
o

3

o
a

S

o

O
03

0

"3

d

CO

50

t— i

H

K

SQ

US

^

—

%

%

%

%

%

%

%

11

96.12

1.72

0.85

0.43

0.42

42

89

0.71

0.60

3-27-09

95

90.84

5.04

0.92

0.48

0.44

58

89

0.70

2.50

3-22-11

90

86.72

7.20

1.23

0.52

0.71

67

129

1.06

3.79

•■

Average

:, 91.16

4.70

1.00

0.48

0.52

52

102

0.83

2.31

Max.,

96.12

7.20

1.23

0.52

0.71

67

129

1.06

3.79

Min.,

86.72

1.72

0.85

0.43

0.42

42

89

0.70

0.60

The moisture content of these sirups varied from 27.91 to
32.56 and averaged 29.93%.

The sucrose showed relatively small variations, ranging from
61.30 to 65.49 and averaging 63.87^ .

The invert sugars tend to run high, an average of 3.29' <
being found with extremes of 1.16 to 5.09%. The fact that

444 Bulletin 167

all the saps were decidedly acid after inoculation with this or-
ganism, would serve to explain the high invert sugar content.
The ash and malic acid value figures are all well above the
Vermont standard.

Group ii. Last run, sweet. These samples were secured
late in each of three sugar seasons, 3 in 1909, 1 in 1910 and 5 in
191 1. The trees were freshly tapped and clean spouts and pails
used. The sap appeared bright and clear and, with one excep-
tion, was boiled down immediately after collection. Hence the
conditions under which these saps were secured may be deemed
to be beyond criticism.

The three samples taken in 1909 displayed exceptionally
fine flavor and color, all grading 1 in flavor and 3. 7, and 5 in
color and scoring 950. 850 and 900 respectively (pages 357-358).
The single sample secured in 1910 and the five obtained in 191 1.
however, while being extremely light colored, had a pronounced
"buddy" flavor, which renders a sirup particularly obnoxious
for food purposes. As maple sirup is usually purchased for
its flavor rather than for its sweetening properties, sirup which
is thus affected is desired by neither manufacturer, dealer, nor
the purchasing public.

"Buddy'' sirup is secured only when a freeze occurs after
the true sugar season has ended and a few warm days have
started the development of the leaf buds. Sap will run on suit-
able days following such freezes, and if the trees are retapped
and clean utensils used, the grade of goods now being considered
will be secured. Should the old tap-holes, spouts and buckets
be used, the material will possess a mixture of "buddy" and
other objectionable flavors, due to the contamination of the
spouts and pails1. This latter condition will be again referred
to in table 32 on page 455 and in the appended discussion.

'In this connection note the statements made on pages 379 and 417
concerning "buddy" flavor and its probable origin, as well as the state-
ment immediately succeeding table 27 on page 446.

Micro-organisms 01? Maple Sac

li;.

TABLE 27. MAPLE SIE1 P. LAS! Bl IS BWEJ l

I

B

t-4
-

A

Ed

01

-
a

a

Oi

CO

CO

od

i

CJ

a

—

<v

<u

A

=

*

u

3

o>

-*— «

o

4-9

— ,

ja

3

•_

—

9

o
o

>

o
o

"o

o

a
o

"3

O
CO

s

cd

-
-

r.

"_

fe

■J2

s

OS

M

—

■/.

*5

<—

%

%

';

%

%

%

%

%

21

6

1

950

34.31

62.53

0.54

0.54

0.33

0.21

0.73

1.3a

24

7

1

s:,(i

29.41

67.75

0.43

0.50

0.33

0.17

0.71

1.20

2G

5

1

900

32.67

64.92

0.34

0.54

0.31

0.23

0.67

0.86

NT

4

5

525

3S.12

60.19

ii. n;

0.49

0.26

0.23

0.61

0.43

122

5

5

500

32.55

64.61

0.45

0.49

0.30

0.19

0.52

1.38

124

:>

5

500

29.35

69.62

0.42

n..",2

0.36

0.16

0.43

0.34

126

6

."i

475

32.71

63.56

0.21

* .

*

*

0.64

• • • •

128

4

5

525

31.81

64.23

0.21

0.40

0.20

0.20

0.56

2.7:i

129

5

:>

500

28.81

67.87

0.08

ii.:, I

0.36

0.18

0.44

2.26

Average

\ 5

3.7

G36

32.20

65.03

0.32

0.50

0.30

0.20

0.58

1.37

Max.,

7

5

950

38.12

69.i 2

ii.:, i

0.54

0.36

0.23

0.73

2.7!i

Min.,

•i

1

475

28.81

60.19

0.08

0.40

0.20

0.16

0.43

.34

MAPLE SIRUP. LAST RUN SWEET

Calculated to a moisture-free basis

c
=

ALKALINITY

0)

3

,<-]

ad

-

w

CO

A

>•

01

Cd

cd

oc

a

49

CO

O

CO

CO

Cd

CO

C3

03
3

a>

eg

4)

2

<u
S

3

cd

0

CD

cd

|

CO

s
-

O

CO

—

a

-7.

—

-f.

—

02

—

*~.

—

-

%

%

%

%

%

%

%

21

95.19

0.82

0.83

0.50

0.33

58

91

1.11

2.05

1-1 2-1 1!»

24

96.02

0.61

0.71

0.47

0.24

54

54

1.01

L.65

> 1

26

96.42

0.51

0.80

0.46

0.34

54

62

1.00

1.27

4-16-09

87

97.27

0.26

0.79

n.41

0.38

68

145

0.98

0.52

t -3-10

122

95.80

0.67

0.73

0.45

0.28

68

59

0.76

2.11 J

4-21-11

124

9S..-.4

0.59

0.74

0.52

0.22

56

47

0.61

0.48

12 1-11

126

94.46

0.31

*

* .

*

*

*

0.95

"

128

94.21

0.31

0.59

0.29

0.30

48

56

D.S2

1.07

1 27-11

129

95.37

0.12

0.75

0.51

0.24

59

4:,

0.61

3.15

1-28-11

Average

95.92

0.47

0.74

0.45

0.29

58

70

0.87

2.00

Max.,

98.54

0.82

0.83

0.52

0.38

68

14.",

1.11

4.<i7

Min..

94.21

0.12

0.59

0.29

n.22

48

45

0.61

.48

''Not determined through inadvertence.

44ti Bulletin 167

The moisture content of these sirups averaged 32.20, the
maximum being 38.12 and the minimum 28.81%.

The sucrose averaged 65.03 with extremes of 60.19 and
69.62%.

The invert sugar was exceptionally small in amount, averag-
ing only 0.32% and varying between 0.08 and 0.54%. This
in itself is proof that care was taken in securing the sap and
that no time was lost before boiling. The low invert sugar,
together with the exceptional light color, also leads to the infer-
ence that the objectionable flavor was not caused by external
contamination, but was due to some change or impurity in the
sap before it left the tree, occasioned by changes occurring inside
the tree, incident to the renewal of the yearly spring functions.

The total ash content runs low in 5 out of 8 samples, the
average on a moisture free basis being 0.74, the maximum 0.83,
and the minimum, 0.59%. The insoluble ash is low in but one
instance, No. 124 being 0.01% deficient. The malic acid, value
meets requirements in every case.

Group 12. Inoculated with fluorescent bacteria and with
spore-bearers. The saps used in securing these samples were inoc-
ulated with various strains of bacteria, mainly of the fluorescent
type. Through inadvertence, spore-bearing organisms of the sub-
tilis type commonly found in soil gained entrance1. The results
were strikingly influenced by the presence and action of these
spore-bearing organisms. The flavor was seriously injured, 4
out of 7 samples grading 4 (poor) and the rest of them 3
(medium). The color was darkened several shades and showed
a large depreciation as compared with the controls, although
none were classed below a Xo. 2, as commercially rated. By
comparison with the results obtained with fluorescent organisms
alone, it appears that the color injury should be attributed
largely to the introduced species, while the intruder is doubt-
less chiefly responsible for the ill flavor and the high invert
sugar content.

1 See statement concerning this matter, page o77.

Micro-organisms or Maple Sap

141

TABLE 28. MAPLE SIRUP. INOCULATED Willi FLUORESCENT BACTERIA \\l>
OTHEB ORGANISMS AND WITH SPORE-BEARERS

Ed

a;

£>

a

cd

i —

a

-*

CO

>

a>

3

U)

a

CO

cd

_

a

s

i>

■j.

Ctj

a>

^

S

—

<u

a>

a

cd

O

a>

■W

o

_

3

3

CJ

B

cd

o

c3

o

2

V

0)

>

03

O

"o

o

CO

a

rt

•a

a

SQ

O

fe

BQ

i«3

02

"-1

E-<

02

g

M

%

%

%

%

%

%

%

%

75

9

o
t>

G75

40.32

50.57

6.43

0.78

0.26

0.52

0.73

1.17

7G

10

o

G50

38.02

53.94

...54

0.80

0.34

0.46

0.76

0.94

77

8

o
d

700

39.G7

53.17

4.97

0.79

0.34

0.45

0.80

0.G0

78

8

4

575

39.98

50.33

6.29

0.74

0.24

0.50

0.71

L.95

79

7

4

GOO

47. GO

41.2S

7.99

0.67

0.19

0.48

0.74

1.72

80

](i

4

525

45.03

43.54

8.81

0.72

0.24

0.48

0.73

1.17

81

7

4

600

38.72

54.68

4.60

0.09

0.34

0.35

0.66

0.65

Ave rag

3, 8.4

3.6

G18

41.33

49.64

6.38

0.74

0.27

0.47

0.73

1.18

Max.,

10

4

700

47.G0

54.68

8.81

0.80

0.34

0.52

II. Ml

1.95

Min..

7

3

525

38.02

41.28

4.G0

0.67

0.19

0.35

0.66

0.60

MAPLE SIRUP. INOCULATED WITH FLUOBESCENT BACTERIA AND OTHER ORGAN-
ISMS AND WITH SPORE-BEARERS

Calculated to a moisture-free basis

Si

a

2

B
od

a

ALKALINITY

O)

"cd

•a

EO

A

r"

o>

od

a

eS

CO

3

M

a

00

cd

OQ

.3

cd

>d

<D

to

OS
ID

4>

—

cd

3

o

cd

a

O

_

X!

3

3

CJ

+j

—<

o>

od

^r

o

p

O

l>

o

O

to

3

©

CO

3

03

0

m

h-l

H

OS

03

r'.

—

od

P

75
76
77
78
79
80
81

%
S4.74
S7.03
88.13
83.86
78.78
79.20
89.23

%

10.77

8.94

8.24

10.4S

15.25

16.03

7.51

Average, 84.42 11.03
Max., 89.23 16.03
Min., 78.78 7.51

%
1.30
1.29
1.31
1.24
1.28
1.30
1.12

1.26
1.31

1.12

%
0.43
0.54
0.56
0.40
0.36
0.43
0.5G

0.46
0.56
0.36

Ye
0.87
0.75
0.75
0.84
0.92
0.87
0.5G

0.80
0.92
0.5G

71
59
G2
61
64
61
G2

63

71
59

118

70

86

169

184

175

50

122

184
50

%

L.22

1.23

1.32

1.18

1.42

1.32

1.07

%
1.97
1.51
1.00
3.24
3.27
2.15
1.07

1.25 2.04
1.42 3.27
1.07 1.00

3-24-10

44s Bulletin T67

The entire lot of 7 samples were a li^lit weight sirup, the
moisture content running- from 47.60 to 38.02 and averaging
41.339? • The sucrose is low owing to the high water and invert
sugar content, averaging but 49.64, with extremes of 41.28 and
54.68%. The invert sugar runs uniformly high and without
the extreme variations noted in some of the previous groups. The
extremes are 4.60 and 8.81 with an average of 6.38^7 , which,
on the moisture free basis, is equivalent to n.03%. The ash and
malic acid value data are well above standard.

Group 13. Inoculated with green molds. The organisms
here used are the ordinary green molds frequently seen on stale
bread. \\ "bile of common occurrence, they do not grow vigor-
ously at low temperatures, hence they are of minor importance
Si > far as maple sap is concerned ; but probably no other single
group of organisms does so much damage to sirup after it^
manufacture. When introduced into the sap. they seriouslv
impaired both the flavor and color of the resulting sirup. 3 out
of the 4 samples grading 4 in flavor, corresponding to poor
quality, while the color showed a depreciation of over 4 points
from the control. The content of invert sugar was also noticea-
bly increased.

r. rt

1

—

>

-
-

■ —

Oj

:f.

t-

*"

~

—

d

r

£

p

-

—

tc

d

-

o

—

-

:l

s

~

0-4

2

o

X

—

>■

-

-

—

w

-.

a

w

—

~

—

^

o

-

-_

>

~

■ —

r-

c

'*«*.

9 10

Plate VIII. — Bacillus aceris. Figures 1-5. Successive photographs of
living organisms during fission. Figure G. Flagella preparations.
Lowet's stain. Figure 7. Stained preparation, gentian violet.
Figure 8. Living organisms showing chains in motion. Figures
9 and 10. Living organisms on agar, showing orientation. (See
pages 480-482.) X1350.

M [CRO ORGANISMS OF M Al'I.i- SAP

I I'.'

TABLE 29. MAPLE SIRUP. INOCULATED Willi GREEN MOLDS

CU

a

-

—

a

—

-i

>

-

a

T.

a

a

.

M

so

to

es

■-

2

=

n

.

h

4>

-

SL

«

-

-

;

01

~

c

u

l-«

.a

CJ

-.—

s

o
3

U

>

o
u
03

o

OS

o

>
a

ed

c

EH

o

X

Z
-

cd

—
a

-

%

%

%

%

%

%

%

%

10

10

4

525

32.8G

64.27

1.75

0.72

0.24

0.48

0.49

-0.09

17

10

2

725

34.08

61.15

2.29

0.72

0.20

0.52

0.95

0.81

111

7

i

coo

28.29

GC.91

3.15

0.55

0.38

0.17

0.48

0.G2

117

9

4

550

30.70

G3.12

2.58

0.51

0.35

0.1G

0.42

2.G7

Average

', 9

3.5

000

31.48

63.8C

2.44

0.C3

0.30

0.33

0.58

1.01

Max.,

10

4

725

34.08

GG.91

3.15

0.72

0.38

ii.r.2

0.95

2.67

Min..

7

2

525

28.29

G1.15

1.75

0.51

0.20

0.1G

0.42 -

0.09

MAPLE SIRUP. INOCULATED Will! GREEN MOLDS

Calculated to a moisture-free basis

HI

E
cd
f.

ALKALINITY

od

—

,C3

>

-

be

BO

a

a

■J.

00

c

00

-4^

DQ

od

cd
o

3

3

3

3

3

ej
od

a

—

4)

=

u

>
a

cd

0

C
so

0

O
H

"3

03

HH

V.

03

—

<<

—

-

1G

17

111

117

% %

95.73 2.61

92.7G 3.47

93.31 4.39

%

1.07
1.09
0.77

% %

0.3G 0.71

0.30 0.79

0.53 0.24

91.09

.72 0.74 0.51 0.2J

Average, 93.21
Max., 95.73
Mm.. 91.09

3.5G
4.39

2.G1

0.92
1.09
0.74

0.44 0.48
0.53 0.79
0.30 0.23

39 140

4G 1C1

Gl 57

G4 52

52

G4
39

%
0.72
1.44
0.67
0.G1

%
-0.13
1.24

ii.s.;
3.84

103

n;i

52

0.86 1.45
1.44 3.84
0.G1 -0.13

4- 1-09

3-27-11
3-30 1 1

The moisture shows less variation than usual, the extremes
being 34.08 and 28.29 and the average 31.48%. The sucrose
averages 63.86, with a minimum of 61.15, and a maximum of
66.01'' . The invert sugar figures run fairly high, the extremes
being 1.75 and 3.15 and the average 2.449? Three green mold
organisms were used in the inoculation of these samples and

450 Bulletin 167

they all appear to exert a marked inverting action on sucrose.
The total and insoluble ashes tog-ether with malic acid value
are above standard in all cases.

Group 14. Tin z's. wooden buckets. The sap from 6 trees
was used in this phase of the work. All tap-holes, spouts and
utensils were made strictly clean and the procedures were iden-
tical in both cases. The sap was concentrated as soon as col-
lected. That obtained in the tin buckets ran 2 days earlier than
that gathered in the wooden buckets, but, as a check, in order
to be certain that the two days' intermission exercised no in-
fluence on the results, bacterial counts were made on sap ob-
tained in tin buckets from the same trees and spouts after the
close of the experiment. It was practically free from bacteria
of any kind; (see page 343). The grade of sirup produced
from the tin buckets (Xo. 66) was of the highest quality, rank-
ing 1 in flavor and 3 in color, and scoring 950. That secured
from the wooden buckets (No. 67), in marked contrast to that
made when tin buckets were employed, ranked only 4 in flavor, 9
in color and scored but a total of 550 points out of a possible
975. Furthermore its invert sugar content was high, there be-
ing seventeen times as much present in the sirup secured in the
wooden buckets as was found in that made when tin buckets were
used. This is doubtless to be attributed to the organisms al-
ready existing in the wooden buckets since, although thoroughly
cleansed, they were not new and were painted on the out side-
only.

Both sirups were light in weight containing respectively
37.78 and 38.33 perccnts of water.

The sucrose is considerably higher in Xo. 00 than in Xo.
67, owing to its low invert sugar content.

The standards for ash and malic acid value are fully met
by both samples.

Micro-organisms of Maple Sap

I.M

TABLE 30.

MAPLE SIKUP. TIN VS. Woom;.\ BUCKETS

.a
a

S

«S
DC

^

**■

~

•^3

:i

^

ft

0)

Gfl

rt

4>

'7"

0

7

o>

—

Pd

.

3

V

fcj

o

i_

^-i

,■•*-

■_

c

.*

h

—

-

r:

~

q

o

5

o
o

a

c

E

~

/.

_

E=l

50

<*.

X

—

H

-z

M

<i

MAPLE SIRUP. TIN vs. WOODEN BUCKETS

Calculated to a moisture-free basis

—

%

%

.%

%

%

%

%

%

66

1

950

37.78

60.12

0.25

0.49

0.31

0.18

0.52

0.84

G7

9

4

550

oo oo
OO.OO

54.42

4.26

0.62

0.34

0.28

0.73

L.64

.a
3

3
3

/:

■f:

O

AI.K.M

I N 1 T V

i .

■J

■r.

fl

<~

V

be

•£

.3

—

m

GO

a

0>

Hi

a

a)

~

£

a

f*

1

-J.

y.

f.

T
-r
a

1

—

-

66

67

'<

96.62

SS.25

% %
0.41 0.78
6.91 1.01

%
0.49
0.55

%
0.29
0.46

65

% %

59 0.. 4 L.35 3-22-10
103 1.19 2.64 3-24-10

452 Bulletin 167

Group 15. Inoculated with pink yeast; burned control.
These two samples, while grouped together, bear no relation to •
each other. The pink yeasts are not as common as are the red
and gray yeasts previously mentioned, but, like them, are en-
countered late in the season. The effect on sirup made from
sap inoculated with pink yeast is shown in No. 98, which may
be considered a typical yeast sirup. Both flavor and color were
impaired, particularly the former. The sirup ranked poor in
quality and, while grading commercially as No. 2, showed a
color depreciation of 6 points. This organism exerted a marked
inverting action on the sucrose, but the other results, including
those for ash and malic acid value, are normal in spite of the
increased concentration, indicated by the low moisture content
of 29.26%.

The burned sample. No. 74, listed in this table, was a control
on the fluorescent and spore-bearer series shown in table 12.
Tt was slightly burned, so that the flavor and color were seri-
ously affected. The burning had but little if any effect on the
invert sugar percentage, as only 0.70$ calculated to the mois-
ture free basis was found.

Micro-organisms of Maple Sap

t53

rABLE 31. MAPLE

SIIU1'.

INOCULATED Willi

PINK yeast;

BUK2S

1 ii ( OB

1 BOL

ty
Xl

a

b

X!

to

o

3

"5

>

5

rt

a

a

3

M

CO

•a

.—

a

a>

<L>

3
to

CO

C3

OS

4)

—

o

at

a

ij

3

CO

-w

—

o

4J

O

i*

~—

A

^h

Ol

a

xa

>

fci

<U

a

3

O

13

o

O

fa

o

y
x/l

O

M
<5

03

a

o
E-i

O
W

to
O

i— <

OS
kH

3
-

%

%

%

% %

%

%

%

98

9

4

550

29.2G

61.33

6.15

0.59 0.37

0.22

0.50

2.17

74

9

4

550

41.40

56.47

0.41

0.73 0.24

0.49

0.77

0.22

MAPLE SIRUP. INOCULATED WITH PINK YEAST ; BURNED CONTKOl

Calculated to a moisture-free basis

u

,3

ALKALINITY

4>

"3

13

x>

B

3

^

A

>

4>

60

xl

id

XI

tt

13

a

a

3

xi

to

cS

V

Sj

1>

S

tu

sj

o

XI

o>

XI

SS

c_

to

■M

O

—

XI

^

XI

^^

a>

a

a?

cj

3

o

o

^M

■O

o

►

to

K

a

3

a

^H

3

o

03

3
i— i

a

P

Q

%

%

%

%

%

%

%

9S

86.70

8.69

0.83

0.52

0.31

62 74

0.71

3.07

3-26-11

74

96.36

0.70

1.24

0.41

0.83

51 99

1.31

0.39

3-24-10

454 Bulletin 167

Group 16. Last run sour. The sirups thus listed were
made from sap which was collected late in the season, and which
was cloudy as it dripped from the spout. The tap-holes, spouts
and buckets were contaminated. In every case but one the sap
was evaporated on the same day it ran.

Referring- to the column headed flavor in the following
table, it will be noted that the three samples. Nos. 22, 23 and 25.
secured late in the season of 1909, graded 3 in flavor, corre-
sponding to medium quality, and 8, 14 and 14 respectively in
color, indicating the second and third grades. No "buddy" sirup
was obtained in t<;oo,. The sirups secured in 1910 and n;ir
graded 5 or 6 in flavor indicating buddiness or worse than bud-
diness. The color in all but Nos. 121 and 125 was seriousb
affected, grading from 11 to 15.

Micro-organisms of Maim.k Sap

l :> ."•

TABLE 32. MAPLE SIRUP. LAST RUN SOUR

p

c

-

**

a

|

;_

■/.

T.

z

Cv

a

ad

a

~*

tl

^

v.

T

P

$

GQ

"7

re

i

~

|

d)

'—

i

ad

■~

—

T.

-

EL

o

ey

s

p

-w

—

3

*—

-_

*Z

E

re
/.

S
o

-

>
ad

0

an

If.

z

M

3
1.

z
a

ad
o

p

©

CO

a

"3

a

%•

%

%

%

%

%

%

%

22

8

3

700

34.45

62.45

1.81

0.64

0.42

0.22

0.87

-0.22

23

14

0

550

34.73

60.41

2.32

0.74

0.40

0.34

0.58

L.22

25

14

3

550

38.68

57.67

1.92

0.86

0.26

0.60

1.15

0.28

88

11

6

250

35.57

60.93

1.15

0.80

0.35

0.45

0.83

0.72

121

7

5

450

35.85

60.26

0.22

0.59

0.25

0.35

0.69

0.39

123

11

6

250

34.01

62.82

0.42

0.54

0.28

0.26

(I.:,:,

1.66

125

7

5

450

35.67

60.39

0.34

0.53

0.27

0.26

0.58

2.49

127

15

6

150

OO.OD

58.25

3.06

0.80

0.32

0.48

ii.s:,

3.6S

130

12

6

225

33.41

62.50

0.89

0.68

0.34

0.34

0.65

1.87

Av'ge,

11

4.8

397

35.08

60.63

1.35

0.68

0.31

0.37

0.75

1.51

Max.,

15

6

700

38. 6S

62.82

3.06

0.86

0.42

0.60

1.15

3.68

Min.,

7

♦ >

150

33.36

57.67

0.22

0.53

0.25

0.22

0.55

0.28

MAPLE SIRUP. LAST RUN SOUR

Calculated to a moisture-free basis

o

ALKALINITY

-

X!

M

K

_-

S

U

y.

-^

r*

z

od

a

m

re

—

a

c

GQ

'r;

re

3J

re

a

T

S

<V

cv

re

u

.z

0!

3

~

T

a

o

i.

~—

^

j^

3

"-

;_

c

£
re
a:

X

0J

re

H

©
v.

k

/.

7.

a
'—

re

%

%

%

%

%

%

%

22

95.27

2.76

0.98

0.64

0.34

85

76

1.:::;

0.34

4-12-09

23

92.56

3.55

1.13

0.61

0.52

58

172

0.90

1.86

••

25

94.05

3.13

1.40

0.42

0.98

98

173

1.87

-0.45

4-l(i-0!t

88

94.56

1.79

1.24

0.54

0.70

52

173

1.29

1.12

4- 3-10

121

93.94

0.34

0.91

0.38

0.53

59

98

L.08

3.73

4-21 1 l

123

95.33

0.64

0.83

0.43

0.40

56

73

0.84

2.3C

4-24-1 1

125

93.88

0.53

0.S2

0.41

0.41

53

94

0.90

3.87

"

127

87.41

4.59

1.20

0.49

0.71

59

144

1.2S

5.52

4-27-11

130

93.86

1.34

1.03

0.52

0.57

71

99

0.98

2.79

4-28 1 1

Averag<

3, 93.41

2.07

1.05

0.48

o.:>7

64

122

1.16

2.31

Max.,

95.33

4.59

1.40

0.64

0.98

98

173

1.S7

5.52

Miu.,

87.41

0.34

0.82

0.3S

0.34

52

73

0.84

0.45

4.~>fi Bulletin 167

The moisture content of these sirups showed less variation
than has been noted in many of the groups. The average was
35.08, with extremes of 38.68 and 33.36^ .

The sucrose averaged 60.63. with extremes of 57.67 and
62.82%.

The invert sugar averaged 1.35%. Nos. 121, 123 and 125
contained but small amounts, which serves to show that in these
cases the infecting organism did not act as an invert sugar
former. The remaining samples, however, show quite a marked
increase, the invert sugar content of No. 127 being 3.06%.

The total and insoluble ash and malic acid value are well
above the standard in ever}- case.

Group 17. Sour sap, kept. The three samples thus listed
were secured in 1910 just previous to the buddy sample No. 88
mentioned in table 32. They represent a composite of the small
runs during several days toward the close of the season. Tap-
holes, spouts and pails were contaminated through the ordinary
natural sources, as was indicated by the cloudy appearance of
the running sap. The flavor in each case was a poor medium.
The color of No. 84 was darker than that of any other sample
secured, with two exceptions grading 20, the extreme limit on
the colorimetric scheme. Nos. 85 and 86 were even darker than
No. 84 and defied grading. An attempt was made during the
boiling process to clarify No. 86 by means of the usual white of
egg treatment which, in this case at least, was ineffective.

M [CRO ORGAN [SMS "!■' M.\ri.K, SAP

i:.7

i'ai'.i.i. :::;. maple sirup, from soi b sap kept

A

cu

«3

■o

a

—*

w

►

O)

.a

cd

a

a

a>

3

S3

CO

cd

V

2

CJ

a

—

o

o>

3

01

o

4->

cd

0)

3

3

cd

^
■*->

a

as

m

o
o

U

cd

o
09

3

w

>

a

a

O

3

O

«2

O

a

"cd

•a

a
P

%

%

%

%

%

%

%

%

84

20

31

350

33.G7

60.45

1.88

0.99

0.34

0.65

0.65

2.3G

85

20+

31

325

3G.G7

59.96

0.99

0.89

0.23

0.G6

0.73

0.7G

8G

20+

325

34.72

60.85

0.81

0.91

0.27

0.64

0.82

1.89

A.v'ge,

20

31

333

35.02

60.42

1.23

0.93

0.28

0.65

0.73

1.1'm

.Max.,

20+

350

36.67

60.85

1.88

0.99

0.34

0.6G

0.82

2.3G

Mill.,

20

325

33.67

59.96

0.81

0.89

0.23

0.64

0.65

0.76

MAPLE SIRUP. FROM SOUR SAP KEPT

Calculated to a moisture-free basis

ED
CO

p

cd
U)

3
cs

>
a

.3.

to
cd

"3
o
fcH

GO

cd
o

.a
cd

—
3
"o

CO

a

ALKALINITY

OJ

3
>

cd

CJ

"3

*— »

■a
a>
a

1

-O

a
P

a

3 '

a

v

a

a

cd
m

a

73

cd

3

3
O

ft

73

cd

0)

3

3
©

a

4)

-4-1

84

91.14

2.83

1.50

0.51

0.99

75

122

0.98

3.55

4- 2-1C

85

94.68

1.57

1.41

0.37

1.04

63

130

1.15

1.19

II

86

93.21

1.23

1.40

0.42

0.99

65

137

1.26

2.90

II

Averag*

i, 93.00

1.88

1.44

0.43

1.01

68

130

1.13

2.55

Max.,

94.68

2.83

1.50

0.51

1.04

75

137

1.26

3.55

Mill.,

91.14

1.23

1.40

0.37

0.99

63

122

0.98

L.19

i58 Bulletin i<>~

The moisture content of the three sirups varied from 36.67
to 33.67, averaging 35.02%.

The sucrose content was quite uniform and averaged 60.42' < .

The invert sugar percentages were 1.88, 0.99 and 0.81.
averaging i.239r. These figures are low enough to indicate
that, although the contaminating organisms were very abundant,
bacterial counts showing over 11,000,000 per cc. they were rela-
tively inactive as producers of invert sugar from sucrose. As has
been stated the color of the sirup was most seriously affected.

Both the total and insoluble ash contents as well as the
malic acid values were above standard.

Summary of Averages Secured on the Sundry Groups Dis-
cussed

Table 34 displays the averages of the individual analyses
of the several sirups examined, in the order previously discussed,
together with the average for the 128 samples. The differences
in physical characteristics and chemical composition are indicated
in the original material and the moisture- free basis portions of
the table.

The color averages j.^. corresponding closely to first grade.
The darkest color was obtained in the samples located in the
"sour sap, kept" group. This group also showed the highest
depreciation from the control as regards color. The lightest
color was secured in samples grouped under the captions "tin
buckets" and "control."

The flavor averages 2.9, corresponding to a quality just be-
low medium. The finest flavor was obtained in samples grouped
under the term "tin buckets" and "control" rating as 1 and 1.4
respectively, the poorest sample in this respect was located in
the group denominated "last run, sour," which included several
buddy sirups. Excluding the samples rating- 5 and 6 in flavor
(buddy) from the color and fla-or averages, the average color
and flavor figures thus revised for the remaining 116 samples are
7.? and 2.6 respectively, equivalent to an average score of 719.

Micro-organisms of Maple Sap

-l.v.i

|Mim(l.l.l|.l|Ml. 1

.iii|i:.\ pi.ii: - > 1 1 ' : 1 V

l|Sl! .I|I|1I|(ISII|

i[su <>i<|ii[<>s

r.

c

l[Sl! IB|<»X

jbSus j.ioaui

aso.i»ns

<

U

■f

CO

3Jll)s;(.iv

3JODg

.IOAU[..>I

jojoo

O

.uqmiiu dnojf)

L"

m ■* ffl W M 00 H C 00 CO t- OH i- ^ f l» M H I- CO
JOlOaNOOOOOOSOiHOKtOHMlOtC t-h
C I— I <3 I— I i— I O t— I r-5 I— I t— i— I r-l i— | O I— I CN O 1-5 1— " r-5

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Micro-organisms of Maple Sap t61

The average moisture content for the entire i_>X samples
is 34.63%. This is practically the moisture percentage of a
standard eleven-pound to the gallon sirup.

The average sucrose figures 61.44%, ancl tne invert sugar
percentage, l.6o%, agree quite closely with the averages, 62.649?
and 1.49%, secured by Bryan in an examination of 395 samples
from all parts of the United States where maple products are
made.

The average total ash figure on a moisture free basis is
0.93%, and extremes are 0.74 and 1.44%. The minimum figures
occur in the "last run, sour" group and include considerable num-
ber of buddy sap samples.

The average insoluble ash percentage is well over the stand-
ard in every group, the minimum being 0.29, the maximum 1.01,
and the average 0.59%.

The malic acid value is likewise above the standard limit,
with a minimum of 0.71, a maximum of 1.21, and an average of
0.90%.

The grand average for the 128 samples secured in the three
sugar seasons is in every particular typical of pure maple sirup,
and would of course more nearly represent the output of thai
particular sugar orchard than would any single sample or minor
group of samples.

INVF.RT SUGAR CONTENT OF MAPLE SIRUP

The invert sugar present in the sirups obtained in this in-
vestigation shows extremes, calculated to a moisture-free basis,
of 0.12 and 28.35%. Invert sugar results from the hydrolysis
or inversion of sucrose, caused by yeasts, molds, bacteria, acids,
etc. Thirty-two sirups, or a quarter part of the entire number of
samples, carried less than 0.60% of invert sugar on a moisture-
free basis, while 57 samples, or 45%, contained less than 1%.
Hence it seems fair to conclude that an invert sugar content in
maple sirup of much more than 1% can only be due to careless
methods in handling or to delay in boiling the sap, or to the sub-
sequent fermentation of the finished product.

462

Bulletin 167

The average invert sugar in the sirups examined, together
with the maximum and minimum amounts found in the indi-
vidual samples, are summarized in table 35 in the order of their

magnitude.

TAI5EE 35.

AVEBAGE INVERT SUGAR CONTENT OF THE DIFFERENT GROUPS AMI
MAXIMUM AM) MINIMUM IN INDIVIDUAL SAMPLES

INVERT SUGAR

Calculated to a
Group Character of moisture-free basis

number organism Average Maximum Minimum

% %

14 Tin buckets 0.41 * *

11 Last run, sweet 0.47 0.82 0.12

5 Failures 0.51 0.90 0.23

1 Control 0.G0 1.58 0.19

15 Burned control 0.70 * *

2 Incubator control 1.04 3.52 0.41

8 Fluorescent 1.09 2.4G 0.38

3 Non-fluorescent 1.52 2.38 0.77

9 Composite 1.64 4.09 0.68

17 Sour sap, kept 1.88 2.83 1.23

16 Last run, sour 2.07 4.59 0.34

4 Pink cocci 2.45 7.90 0.65

6 Red yeasts 3.18 7.63 1.01

1 :: Green molds 3.56 4.39 2.61

10 Bacillus aceris 4.70 7.20 1.72

7 Gray yeasts 6.86 28.35 1 .21

14 Wooden buckets 6.91 * *

1 5 Pink yeasts 8.69 * *

12 Fluorescent bacteria and spore-

bearers 1 1.03 16.03 7.51

* Single sample.

It is readily seen that the average invert sugar figures show
a fairly regular gradation from 0.41 to 11.03%, nut tnat tne maxi~
mum and minimum figures among the different groups exhibit
wide variations. Thus the average for group 7, gray yeasts, is
6.869? • Dut u is obtained by averaging eight results, which var\
all the way from 1.21 to 28.35%. These differences in the maxi-
mum and minimum figures found in the same group are doubt-
less due to the fact that certain strains produced a more com-
plete infection and were better inverters of sucrose than were
others. Certain of the organisms used, notably those of the
fluorescenl group, had but little effect on the sucrose. They

Micro-organisms of Maple Sap t63

apparently feed mainly on the proteids and mineral salts and exert.
a detrimental influence on color and flavor. Generally speaking,

the yeasts and molds, which often but not always thrive well
in a slightly aeid medium, together with the spore-bearing bac-
teria, had the most pronounced inverting action on sucrose, either
through the production of invertase, or by the formation of
acid, or both. In many cases they likewise seriously affected
color and flavor.

Most of the remaining bacteria used in this work did not
prove particularly active as invert sugar makers, but their harm-
ful effect was in many instances manifested by the color ami
flavor of the sirup.

Discission op tiik Total and Insoluble Ash and Malic

Acid Values

A survey of the analytical data given in tables 16 to 34 shows
that the ash and malic acid values of 34 of the samples examined
were slightly below the standards used in determining the purity
of maple products. These deficiencies have been noted in the
discussion of the several groups. The question now arises
whether these abnormalities, which in many cases are very slight,
are due :

(1) To the exceptional conditions obtaining in these ex-
perimental trials of the manufacture of sirup from sap, e. g..
the small amounts of sap evaporated, the small number of trees
contributing to the individual samples, and the variation in den-
sit} of the resulting sirup.

(2) To the treatment of the sirups after manufacture
and previous to analysis.

(3) To the effect of the inoculating organisms on the
physical characteristics and chemical composition of the sirups.

These considerations have been clearly explained in the
preceding pages, but their connection with the analytical data
under discussion has not been traced.

For the purpose of easy reference and to assign, if possible.
a definite reason for these failures, all samples showing deli-

464

Bulletin 167

ciencies in ash or malic acid values arc listed by groups in the
following tabic.

TABLE 36. SIRUPS DEFICIENT IN TOTAL ASH, INSOLUBLE ASH, OK MALIC

ACID VALUE

o>

3

o

^2

.a

a

X

o

ai

3
m

>

CALCULATED TO
MOISTURE-FREE BASIS

ja

DS

.^

m

sl

0

cj

a

a>

,a

05 a)

3

od

0
H

0 —

"3

--

a rt

— es
si "■

1 Control, 27

58
G8
89
97
113

2 Inc. cont'l, 9G

" " 104

112

" " 120

4 P'k cocci, 107

« « 31

5 Failures, 10G

108

G R. yeasts, 114

99

7 G. yeasts, 110

115

8 Flu. bac, 102

103

101

100

91

93

94

11G

118

119

9 Comp'te,

11

Last run,
sweet,

13

24
122
124
128
129
117

G. molds,

Average,

Maximum,

Minimum,

34. 4G
36.30

37.33

29.9G

30.3G

29.99

27.81

29. 4G

31.34

38.97

27.90

38.21

30.48

29. G4

30.G9

28.11

30.94

31.04

28.81

28.75

28.3G

27. 7G

26.75

28.06

28.51

29.89

29.30

28.75

29.41
32.55
29.35
31.81

28.81
30.70

30.62
38.97
26.75

63.04
61.80
G0.73
67.11
66.88
67.89
68.82
68.42
67.01
58.06
68.15
59.70
67.64
67.10
66.75
68.17
67.05
65.61
68.11
67.97
69.10
69.59
69.40
68.65
67.69
67.04
67.66
68.59

• 17.7.1
64.61
G9.G2
64.23

Im.ST

63.12

66.50
69.62

:,x.or,

of

/o

0.20
0.28
0.32
0.24
0.13
0.43
0.32
0.29
0.76
0.35
0.50
0.19
0.46
0.63
0.70
1.47
1.20
1.05
0.81
0.96
0.67
0.67
0.29
0.31
0.37
0.48
0.69
0.58

0.43
0.45
0.42
0.21
0.08
2.5S

0.57
2.58
0.08

%
0.75
0.73
0.68
0.61
0.68
0.75
0.71
0.69
0.71
0.71
0.71
1.14
0.69
0.C5
0.71
0.75
0.67
0.72
0.82
0.73
0.71
0.79
0.72
0.83
0.72
0.86
0.84
0.64

0.71
0.73
0.74
0.59
0.75
0.74

0.74
1.14

%
0.53
0.43
0.45
0.32
0.43
0.50
0.46
0.47
0.50
0.45
0.50
0.55
0.47
0.45
0.49
0.44
0.44
0.51
0.58
0.45
0.44
0.54
0.44
0.56
0.41
0.65
0.59
0.42

0.47
0.45
0.52
0.29
0.51
0.51

0.48
0.65
0.29

%
0.22
0.30
0.23
0.29
0.25
0.25
0.25
0.22
0.21
0.26
0.21
0.59
0.22
0.20
0.22
0.31
0.23
0.21
0.24
0.28
0.27
0.25
0.28
0.27
0.31
0.21
0.25
0.22

0.24

,1.2s

0.22
0.30
0.24
0.23

0.26

0.59

0.20

%
0.73
0.83
0.81
1.01
0.73
0.73
0.65
0.60
0.44
0.72
0.58
0.55
0.59
0.49
0.56
0.67
0.65
0.62
0.50
0.60
0.53
0.55
0.90
0.54
0.64
0.65
0.58
0.59

1.01
0.76
0.61
0.82
0.61
0.61

0.66
1.01

0.44

Micro-organisms oi? Maple Sap t65

This table, studied in conjunction with its predecessors,

shows :

(i) That the color averaged 6, with extremes of 3 and
9, indicating that in all cases the goods were of high grade.

(2) That the flavor ranged from 1 to 5 with an average of
3, which corresponds to medium. Of the 34 samples listed, 4
scored 1 in flavor, 9 scored 2, 7 scored 3, 10 scored 4, and .|
scored 5 (buddy). In other words 20 samples or 3 out of 5
•Traded medium or better than medium.

(3) That 10 of the 34 samples were "controls," that 5 were
"last run sap, sweet," that 1 was inoculated with green mold, '1
with yeasts, 8 with bacteria, and 3 with mixtures of yeasts and
bacteria. Hence, 15, or 44% of the total number, were not
artificially inoculated.

(4) That invert sugar was not formed in excessive
amounts.

(5) That most of the samples contained far less water
than the 35% which the standard 11 pounds to the gallon sirup
carries. They were, consequently, considerably heavier than
necessary. But 4 samples contained more than 35%' of water,
while thirty carried from 26.75 to 34.46%, and twenty less
than 30% of water.

The standards of ash and malic acid used were predicated on
an eleven-pound gallon, or 35% moisture, basis. It is quite evident
that the over-concentration secured in these samples together
with their thorough clarification before analysis, resulted in
an increased precipitation and sedimentation of the "niter,"
greater than would have occurred had the sap sirup been con-
centrated only to the standard density. This tended to lower the
ash and malic acid contents of the sample as analysed. This is
strikingly brought out by a comparison of the analyses of samples
containing over 34% water with those containing less than this
amount as shown on the next page.

4f.fi Bulletin i6y

Calculated to a moisture-free basis

Average,

Average above 34% water, 84

Average below 34% water, 42

Average below 30% water, 25

In-

Malic

No. of

Moist-

Total

Soluble

soluble

acid

samples

ure

ash

ash

ash

value

128

34.63

0.93

0.44

0.49

0.90

;r, 84

36.86

1.02

0.45

0.57

1.00

;r, 42

30.05

0.80

0.48

0.32

0.71

;r, 25

28.74

0.77

0.49

0.28

0.66

A gradual drop in tiie total and insoluble ash contents and
in malic acid value occurs as the concentration increases. If
averages show this condition, it is to he expected that the individ-
ual samples, which, in the instances under consideration, rep-
resent the sap from but a few trees and not a composite from
the orchard, would show similar variations, some of which must
of necessity be below the standard of comparison, which, of
course, is based on commercial samples and not on such extreme
conditions as obtained in this investigation.

In judging the purity of maple sirup, it is essential that the
analyst should clearly understand the nature and significance of
the data secured. The standard under consideration includes
the total and insoluble ash contents and the malic acid value.
They represent inorganic and organic constituents respectively.
The slight departure of any one constituent from the standard
should not in itself be interpreted too strictly, particularly if
it be the total ash or malic acid value.

The number of samples below the standard, and the average,
maximum and minimum deficiencies are stated in the following
table.

Micro-organisms of Maple Sap

4<;t

TABLE 37. NUMBER OF SAMPLES AND PERCEN1 BELOW STANDARD l.\ TOTAL
\sll. INSOLUBLE ASH ami MALIC ACID VAL1 I

CALCULATED TO MOISTUBE-FUEE T.AS1S

B«o

Tola! ash

Insoluble ash

Malic acid pa

1'erceut be

low

Percent below

IViv.-m Im

/

£

"

1-

M

~

1-

0)

g

E

CI

01

a

0

—

V

a

z

3

£3

fe

it

s

—

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c

•/>.

.^

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-—

a

<

<<

."-.

X

<

rr.

<,

<

<-.

Control,
Inc. con.,
Non-fluo.,
P'k cocci,
Failures,
R. yeasts,
G. yeasts,
Fluores't,
Comp'te,
B. aceris,
Last run,

sweet, 5
Fluor, bac.
and spore-
bearers, 0
G. molds,
T. buck's
W. buck's
P. yeasts,
Burned
control,
Last run,
sour,
Sour sap,
kept.

.07
.07

.06
.10
.04
.07
.05
.13

.03

16

OS

12

06
10

in;

.is

.02

.06

1

.02

08

2

.02

02

1

.01

05

1

.02

04

0

.02

1
2
0
1
2
1
1
0
2
0

o
0
II
0

I)

Total, 28

0

ii

0

1]

01
01

III

.01

02

03

02

.hi

.01

.01

II

• ■ •

. . .

1

.16

. . .

0

...

1

02

9

o

06

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.ill

1

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• • •

. . .

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• . .

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4

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2

01

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(1

, .

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II
II
II

II

II
o
ii

12

468

Bulletin 167

Twenty-eight samples were deficient in total ash in amounts
varying from 0.02 to 0.18%. The deficiencies mainly occur
among samples in the control groups. Eleven samples were
low in insoluble ash, but the shortages were very slight, varying
from 0.01 to 0.03%, the latter figure occurring but once. There
were 12 deficiencies in malic acid values, many of them being very
small and ranging from 0.01 to 0.16%.

Inquiring still farther into these deficiencies, it will be
found that in several cases but a single item of the three-fold
standard is affected. Eliminating the groups exhibiting no de-
partures from standard, the following table appears.

TABLE 38. NUMBER AND XATUKE OF DEFICIENCIES

a

a

CJ

Character of
organism

c

a>

•a

■a

a —

0 0

01

-

0

0

" v.

n

in insolubl
li and mall
id value

"3

c

-4->

0

CO

a

a

in malic
lue

— -
So

=
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in total
(1 malic
lue

b

> ■
1* Bj

- -

s =

> C es
5 « >

' s a

0

O

0

£

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►J

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■J

►J

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a — !z

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** s S

c o s

- m"C-

> e c «

c

1
2
4
5
6
7
8
;i
11

13

Control

Incubator control

Pink cocci

Failures

Red yeasts

Gray yeasts

Fluorescent

Composite

Last run, sweet .
Green mold

G
4
1
2
2
2
4
1
5
1

1

2

1
9

1
1
0
2
1
0

1
1
0
0
0

1

0
0

1

0

0
0
0
0
0
0

1

0
0
0

0
0
0
0
0

0

II
II
0

II

Totals 28 11 12

Assuming that standard maintenance in two instances and
a very close approach thereto in the third would suffice to pass
a sample, a large share of the samples heretofore listed as
below standard are eliminated and attention is fixed upon the 1 1
lots, listed in the last four columns, four of which are below in
both total and insoluble ash, one low in both total ash and malic
arid value, while six are low in all three items.

Micro-organisms of Maple Sap

h;:»

The following table has been prepared as an aid in locating
the samples that show these deficiencies. The sample numbers
correspond to those hitherto employed pages 352 to 390, refer-
ence to which will enable anyone interested to ascertain the en-
tire history and chemical analysis of each lot.

TABLE 39. DEFICIENCIES GROUPED ACCORDING TO SAMPLE NU.MBKKS

OJ

0

a

0)

"3
cd

c a

p

OS ri OS

a

Z '*

"5

"o

c

_ CS

K -
^ —

0 H OJ

to r

S -■-=

a
c.

B

©

0

0
c

a
> ">

"5

a

£3
So

DQ

1-2

•, a cs

g es >

> tn cj

£ K rt
O

C XI OS

2o„E

> C = 0:
g-cs ►

6

t-^

t— 1

j

j

1-1

j

J

i Control, \

27, 28, 68
89, 97,113

27

27

2 Incubator

\ 90,104,

control,

(112,120

104, 112

112

104

112

4 Pink cocci

107

107

107

107

5 Failures,

106,108

106,108

31,106
108

108, 100

G Red yeasts,

114, 99

114

114

, B

, ,

114

7 Gray yeasts

110, 115

115

. ,

115

. .

t ,

8 Fluores-

cent bac-

j 103, 101

101,102

101

teria,

\ 91, 94

100, 93

9 Composite,

119

119,116

119,118

, ,

. t

1 L9

11 Last run,

S 24, 122, 124

124

, .

124

, .

, B

sweet,

I 128, 129

. .

, .

. ,

, t

Green molds

, 117

Examining closely into the deficiencies of the n. samples
previously mentioned, it appears that Nos. 27, 104, 115 and 124
were more concentrated than the standard requires ; yet, the de-
ficiencies in total ash are but 0.02, 0.08, 0.05 and 0.03% re-
spectively. In three cases the insoluble ash is but 0.01% and in
the other but 0.02% below. They equal or exceed the standard
in malic acid value.

No. 101 contains 28.36% water and is 0.06% low in total
ash and 0.07% low in malic acid value, while, on the other hand,
it is 0.04% over standard in insoluble ash. The remaining six
samples Nos. 112, 107, 106, 108, 114, and 119, fall short in all

47<> Bulletin 167

three items, the total ash ranging- from 0.06% to 0.13%, the in-
soluble ash from 0.01% to 0.03% and malic acid value from
0.01% to 0.16% below standard requirements.

Explanatory of the deficiencies enumerated above, atten-
tion is now called to a procedure that was employed on 11 of the
samples reported in table 36, in which are listed the samples
below standard in any particular, and which, fortunately, included
5 of those now being considered. These 11 samples were
selected at random before the previous compilations had been
made and their choice, at that time, depended on the size of the
sample available, together with the concentration as indicated
by the moisture content.

The treatment to which they were submitted is known as the
"boiling and filtering process" first suggested by tbe writer in
1905. L This, as was then pointed out, is an important procedure
in the certain determination of the purity of a maple product.
Suitable portions of water were added and the samples con-
taining both sirup and precipitated "niter" were mixed, heated
in a water bath at 65 to 75 ° C. for an hour, and allowed to settle
for two days. A portion was then decanted and boiled in a
beaker, until a thermometer inserted in the sirup indicated 1040
C. The sirup was immediately filtered hot through double filter
papers and the ash and malic acid value determined on the clear
filtrate. The results obtained by this procedure are given in the
following table together with tbose originally secured on the
concentrated and clarified samples.

1 Vt. Sta. Rpt. IS, p. 328 (1905).

Micro-organisms of Maple Sap

171

rABLE 40. EFFECT OF DILUTING, BOILING AND FILTERING OH CONCENTRATED
MAPLE SIRUP COMPARED WITH THE ORIGINAL ANALYSES "I THE

SAME SAMPLES

i AM !t,ATED TO A MOISTURE FREE BASIS

•—

1

4-9

OQ

r'.

i.

a

Q

o

r.

a
o

.W.KAI.IMTY

«

. £

S3

3 '-

1

1*5

%

'.

'■>

' ■

Control,

III.-,

B. & F. *

39.21

0.81

o.5:;

0.28

62

05

0.63

"

«

Original

30.53

0.79

0.54

• 1.25

58

48

0.61

a

113

B. & F.

37.25

0.96

0.55

0.41

64

51

(•.75

a

CI

Original

29.99

0.75

0.50

0.25

02

58

0.7::

In. con 1 10

, 104

B. & F.

35. 40

0.77

0.4::

0.34

02

59

0.74

"

II

Original

29.46

0.69

0.47

0.22

62

42

0.60

Pink cocci,

107

B. & F.

35.05

0.79

0.54

0.25

52

55

0.01

CI

"

Original

27.90

0.71

0.50

0.21

57

::4

0.58

Failures,

108

B. & F.

33.27

0.75

0.52

0.2::

54

12

H.52

II

"

Original

29.64

0.65

0.45

0.20

58

::7

0.49

Red yeast*

3, 114

B. & F.

35. 7! '<

0.92

0.65

0.27

58

53

0.62

It

II

Original

30.69

0.71

0.49

0.22

01

52

0.50

Gray yeast

3, 110

B. & F.

35.62

0.81

0.56

0.25

56

59

0.01

II

"

Original

30.94

0.67

0.44

0.2::

58

52

(.. 05

a

115

B. & F.

38.00

0.87

0.61

0.26

65

58

0.00

a

tt

Original

31.04

0.72

0.51

0.21

61

5::

0.62

Flno. bac.

91

B. & F.

35.95

0.78

0.51

0.27

59

59

0.61

"

it

Original

26.75

0.72

0.44

0.28

58

58

0.90

Gr. molds.

111

B. & F.

39.40

0.86

0.60

0.26

59

74

0.00

"

tt

Original

28.29

0.77

0.5::

0.24

01

57

0.67

a

117

B. & F.

35.75

o.S7

0.5::

0.34

62

65

0.64

«

"

Original

30.70

0.74

0.51

0.2::

64

52

o.Ol

*Boile

d and

filtered.

The tabic shows conclusively that by simply diluting ami
boiling the entire sirup, including its niter, to its normal density,
standards are met in every case. All the samples thus treated
show an increased total ash content, sufficient to enable them \<>
meet the standard requirement, save in the case of No. 108.
the moisture content of which when boiled and filtered, was only
33.27%. The insoluble ash percentage has been increased in
everv case sufficient to fullv meet the standard.

472 Bulletin 167

This treatment resulted in an increase in total ash for the
11 samples of 1.27%, averaging 0.115'/ for each sample, with
extremes from 0.02 to 0.21%. The insoluble ash was increased
0.62%, averaging 0.056^, with extremes from — 0.01 to +0.16%.
The soluble ash was increased 0.55%, averaging 0.05%, with
extremes from ■ — 0.04 to -j-0.16%. The standard is met as far as
the malic acid value is concerned in all but one sample (No. 108)
and even in this case the deficiency is slight.

This procedure seems to make it clear that the failure of
several samples under discussion to meet standard require-
ments in certain particulars is not due to the influence of the
inoculating organism employed, but rather to the over-concen-
tration of the sample which, during the long period of sedi-
mentation, caused a larger amount of niter to form and to
settle out than would have formed and settled had the concen-
tration been less and nearer that of the eleven-pound gallon.

Micro-organisms of Maple Sap IT-

. Summary

i. All inoculating- organisms used ( with a single ex-
ception, sirup number 12) had previously been obtained from
'maple sap and the so-called natural infection due to carel<
methods of gathering and handling may result in the introduction
of any or all of the organisms employed in this investigation,
with similar results as regards quality of product.

2. The conditions are more favorable to bacterial, yeast and
mold contamination in the sap toward the close of the sugar
season than earlier because of the higher temperatures, bare
ground, rain, interrupted runs, less cleanly utensils, etc., which
then obtain.

3. The relation of cleanliness in all operations and of
promptness in collecting and boiling the sap to the maintenance
of a high quality of product are strikingly shown by the results
secured in this investigation.

4. The several forms of micro-organisms used in this study
exerted apparently little or no effect on the ash content or the
malic acid value of the sirups.

5. Slightly low ash contents and malic acid values were
often obtained on individual samples of the better grades <>t
sirup, which, however, represented the product from but a few
trees. They occurred mainly when increased concentration be
yond the eleven-pound gallon standard was followed by a pro
longed sedimentation or thorough clarification.

6. The deficiencies noted in ash contents or malic acid
values are not extreme and are due wholly to exceptional con-
ditions of manufacture which would not obtain in commercial
practice.

7. Standard maple syrup should weigh 11 pounds to the
gallon, should carry between 34 and 35$ of water, should give
a Baume reading of 35>4 to 36 at 6oG F., and should contain,
calculated to a moisture-free basis, a total (maple syrup) ash of
0.77%, an insoluble ash of 0.23$ and a malic acid value ol

474 Bulletin 167

0.60'/ . These figures constitute the standard now in use for
determining purity. They are none too low, should be con-
sidered collectively, and, when properly interpreted, should
enable certain differentiation between pure and adulterated
maple products.

Micro-organisms of Maple Sap t75

PART 111
TECHNICAL DESCRIPTION OF CERTAIN BACTERIA OCCURRING

IN MAPLE SAP

By II. A. Edson and C. W. Carpenter

(A.) DESCRIPTION OF BACILLUS ACERIS
(new species)

Summary oe Characters

I. Occurrence and Character

The bacillus occurs as the causal organism in a certain type
of stringy maple sap. in which it produces an acid reaction and
milky appearance. It seriously affects the clearness and flavor of
sirup and also causes an increase in the content of invert sugar.

II. Morphology

1. Form. — A bacillus, with rounded ends, occurring- singl)
or in chains.

2. Size: — .9 to 1 micron by 1.5 to 3 micron-.

3. Agar hanging block. — Organisms occur as single rods or
in long chains according to the amount of moisture present.
Orientation of chains either parallel or irregular.

4. Endospores. — Xot found.

5. Motility. — Very actively motile in liquid media and fre-
quently also on solid media. Two to 7 peritrichiate flagella
readily demonstrated by either Lowit's. Loefflers or the Pitfield
method.

6. Capsule. — Slight capsulation when grown in maple sap,
milk or on certain carbohydrate agars.

7. Involution forms. — Vacuolation occasionally observed on
potato.

8. Staining reactions. — Organisms stained readily with [-10
watery fuchsin. gentian violet, carbol fuchsin, and Loeffler's al-
kaline methylene blue. Not stained by Gram's method.

47<5 Bulletin 167

III. Cultural Features

1 . Agar.

Stroke. — Moderate, filiform to echinulate.

Stab. — Filiform, becoming- villous or plumose, surface growth
abundant, often spreading

Plate. — Colonies round or slightly irregular, smooth becom-
ing radiate or striate, convex, edge entire, undulate or lobate ; in-
ternal structure granular to grumose.

2. Gelatin.

Stab. — Beaded, becoming villous and plumose. Liquefac-
tion napiform, becoming infundibuliform, beginning in 18 days,
complete in from 60 to 80 days.

Plate.— Colonies round or irregular, convex, edge entire
undulate or lobate. liquefaction absent.

3. Nutrient broth. — Strong transient clouding with pellicle
formation. Sediment flaky or membranous, later viscid on agita-
tion.

4. Cooked potato. — Moderate persistent growth, echinulate
or spreading below, convex, glistening, contoured, slimy to buty-
rous. Odor of alcohol. Medium grayed.

5. Milk. — Acid, coagulation delayed, beginning in from 4
to 6 days. Coagulum not peptonized. Litmus milk first red-
dened, then reduced.

6. Starch jelly. — Growth copious, diastasic action feeble or
absent. Reducing sugars not found, but alcohol formed.

7. Silicate jelly containing Fermi's solution. — Scanty
growth.

8. Cohn's solution. — Xo growth.

9. Uschinsky solution. — Growth copious, the fluid becom-
ing pronouncedly viscid.

10. Sodium chlorid in bouillon. — Growth in concentrations
up to and including gc,c . but not in 10$ .

11. Bouillon over chloroform. — Growth unrestricted.

12. Nitrogen. — Obtained from peptone and asparagin.

Micro-organisms of Maple Sap 177

IV. Physical axd Biochemical Features

i. Gas production. — Gas production in maple sap and in
bouillon containing dextrose, sucrose, lactose, maltose, mannit,
and potato extract, but not in bouillon containing glycerin. Gas
composed of carbon dioxid and hydrogen.

2. Growth in closed arm. — Occurred in fermentation tubes
of bouillon containing dextrose, sucrose, lactose, maltose, and
mannit, and in tubes of potato extract, but not in tubes of bouill. >n
containing glycerin.

3. Acid production. — Acid formed in maple sap, and in
bouillon containing dextrose, sucrose, lactose, maltose, glycerin
and mannit.

4. Ammonia production. — Moderate in bouillon.

5. Nitrate reduction. — Nitrates reduced to nitrite-.

6. Indol and phenol production. — Feeble to moderate indol
production in bouillon and in Dunham's solution. Phenol pro-
duction negative.

7. Toleration of acids. — Medium growth in bouillon acidified
with HC1 to -f-25 Fuller's scale and in bouillon acidified to -4- 20
with acetic acid.

8. Toleration of sodium hydroxid. — No growth in tubes
of bouillon having a reaction more alkaline than — 5 Fuller's scale.
The organism was not killed in tubes having an initial reaction
of — 25, since, after twelve days, when the alkali was neutralized
by atmospheric carbon dioxid, fair growth developed in such
tubes. A more alkaline initial reaction invariably killed the
organism.

9. Optimum reaction. — For growth in bouillon the opti-
mum reaction was found to be +10 Fuller's scale.

10. Vitality on culture media. — Transfers from old cultures
on various media, even when these had dried down, developed
promptly.

11. 'Temperature relations. — Thermal death point in bouil-
lon (10 minutes' exposure in water bath in thin walled tubes!

4:78 Bulletin 167

approximately 500 C. Optimum temperature 250 C. Maximum
temperature 370 C. Minimum temperature not determined.
Growth slow at io° C.

12. Desiccation. — Growth occurred after drying for nine
days in some cases, but not after drying for 20 days.

13. Insolation. — Exposure on ice to direct sunlight at noon
in August ion fifteen minutes killed 46.7^ of the organisms.

14. Acids produced. — Not identified.

15. Alkalies produced. — Ammonia.

16. Alcohols produced. — Ethyl alcohol.

17. Ferments produced — The organism digests gelatin
slowly: yet notwithstanding all attempts to demonstrate pro-
teolytic ferments by the milk serum method gave negative re-
sults. Potato starch was evidently acted upon feebly, alcohol
being produced, but diastatic ferments could not be demonstrated.
Invertase formed in very small quantities.

18. Effect of germicides. — Formalin and phenol were tested
in varying: amounts in bouillon. Phenol retarded growth in
dilutions of 1-1000 and killed the bacillus in dilutions of 1-500.
Formalin retarded growth in dilutions of 1-2750 and inhibited
growth in dilutions of 1-2200.

19. Number. — According to the numerical classification of
the Descriptive Chart of the Society of American Bacteriologists
the organism is Bacillus 221. 11 13022.

Detailed Descriptk in
occurrence

At various times during the progress of the study of the
bacteria of maple sap. stringy or ropy specimens of the material
were encountered. These were of two types. The more common
form was usually observed only after the close of the commercial
season and was associated with the presence of filamentous fungi,
yeasts, and various bacteria growing together. This sap always
presented a more or less lumpy appearance when poured from the
bucket. The other type of stringy sap was of a more uniform

Micro-organisms of Maple Sap 4<l»

consistency and was found only a few times during the progress

of the studies.

Such a specimen of sap was first received in the laboratory
during the sugar season of [908. It possessed a decidedly

stringy character, being so ropy that after turning a little of ii
from the Mask in which it was received the material continued to
siphon from the container, even when its mouth was turned up
so as to he somewhat above the level of the sap within. The
material was uniform in consistency, milky in color, and possessed
a pronounced yeasty odor. Examination under the microscope
revealed enormous numbers of actively motile bacteria apparently
in almost pure culture. When plates were poured almost all the
colonies developing were of one type. Cultures obtained from these
were found capable of reproducing the characters of the original
material when introduced into fresh maple sap. As explained
under the head "Cultural Characters," page 403, the stringy
property developed to a pronounced degree only in unsterilized
sap, but since it always appeared after inoculation with this or-
ganism, and only when this organism was employed, and since
the bacillus was always recovered from the inoculated material in
practically pure cultures, there can be no doubt of its causal rela
tion to the condition of the sap.

The organism has a very detrimental influence upon the
quality of sirup. While the color is not always seriously impaired
the sirup is made more or less cloudy, a very unnatural and 1111
pleasant flavor is developed, and the amount of invert sugar i^
increased by the action of the organism.

FORM
The organism is a bacillus with rounded ends, occurring
singly, in pairs, or often in filaments in young agar or nutrient
broth cultures at 20-250 C. Short chains 1 5 to 7 segments) often
occur in cultures 24 hours old and chains of 20 or more segments
are not infrequent. The tendency to chain formation is less pro
nounced in the older cultures and persists longer upon solid media
than in liquid cultures.

480 Bulletin 167

Morphological Characters
dimensions
Stained specimens for measurement were prepared from
young- (24 hour) agar and broth cultures with watery solutions
of the common anilin stains, with anilin water gentian violet, and
with carbol fuchsin. Measurements using a Zeiss homogenous
1 -12 objective and a number 3 micrometer ocular, or by photog-
raphy, showed a diameter of from .9 micron to 1 micron, the
majority being slightly less than 1 micron. The length varied
from 1.5 microns to 3 microns. Living organisms upon agar
hanging blocks meacured by either method were found to have
the same dimensions as the stained organisms with the possible
exception of a few instances, in which there was an apparent
length of 5 to 6 microns. Since careful focusing in some of these
cases revealed indistinct lines of fission it is possible that the
longer bodies are really filaments of 2 or more segments. (See
I 'late Y11I, figs. 9 and 10).

CULTURES ON AGAR HANGING BLOCKS

Agar hanging block cultures were prepared from small blocks
of nutrient agar cut from poured plates and placed on sterile
cover glasses, the surface next to the cover glasses being first
touched with a dilution from a young nutrient broth culture. The
cover glasses were placed on hollow ground slides and sealed
cither with vaseline or with a bit of melted agar to prevent evapo-
ration, and the preparation held at room temperature (20-240 C.)
for observation under the microscope. In abundant moisture the
grouping was similar to that described on page 470 under the
heading "Form." In cultures too dry to afford easy locomotion,
but not dry enough altogether to inhibit motion, long, variously
oriented, vibrating or squirming chains were observed. (See
Plate VIII, figure 8, in which the motion of the chains gave the
negative a badly blurred appearance). Blocks in which motility
was prevented by lack of moisture developed chains of from 2 t<>
many segments, the elements usually showing parallel orienta
tion bnl sometimes developing an irregular grouping.

Plate IX. — Gelatin colonies of Barillas aceris. Figures 1. 2, 3, and i.
Photograph of the same colony one. two. lour, and seven days old.
Figure 5. Colony seven days old. showing transition stage from
entire to lobate edge. (See page 4ST.) X 75.

/

Plate X. — Flagella preparations from 24-hour agar slants ( Loeffler's

stain l. Figs. 1-4 show capsulation.

(See

Fig. 1. B. aceris

Fig. 2.

B. parallelus

Fig. 3. B. parallelus

Fig. 4.

B. parallelus

Fig. 5. Ps. fluoreseens,

Fig. 6.

Ps. fluoreseens,

strain CXLV

strain CXLV

pages 483, 554-556.) X

1 500.

Micro-organisms of Maple Sap t81

FISSION

The first indication of fission observed was a slight constric-
tion at the center of organisms which were then 3 microns in
length. The constrictions hecame gradually more pronounced
and in the course of a few minutes two daughter cells each 1.5
microns in length were produced. These sometimes remained
attached indefinitely while in other cases they separated almost
at once. In the specimens observed the daughter cells showed no
increase in length for a period varying from 20 minutes to 2
hours after division. There then occurred a period of rapid
growth followed immediately by fission. The process of elonga-
tion and division were observed to take place in periods of from
20 to 40 minutes. Fission was completed in from 5 to 8 minul
after the first indication of its occurrence was observed. The
active motility of this organism in abundant moisture made it
impossible to carry out these studies under conditions of optimum
humidity. The colonies under observation were so dry thai
growth continued only a few generations, and it is fair to assume
that the rate of growth observed is far below the maximum.
( Plate YIN, figures 1 to 5).

GROUPIXG

The formation of chains and filaments has been noted.
Pseudo-zoogloea masses were observed in young cultures on
broth, maple sap, and other liquid media.

MOTILITY AND IXACKU.A

Active motility occurred in young cultures in all liquid media
employed. Colonies upon freshly poured plates of agar and
gelatin exhibited internal motility under a Zeiss A objective and
number 4 ocular. Motility became less marked in older culture-
hut was seldom entirely absent even in preparations made from
cultures several weeks old, whether from liquid or from solid
media. Flagella stains were obtained by Lowit's, Loeftler's. and
the Pitfield methods, in preparations made from diluted con
densation writer of 24-hour old agar slant culture-. These

482 Bulletin 167

showed each rod to have from 2 to 7 peritrichiate flagella fre-
quently developing a length of 15 microns. (Plate VIII, fig-
ure 6).

SPORES

No indications of spores were observed although search was
made both by microscopic methods and by means of thermal
death point determinations in old cultures upon agar and upon
cooked potato.

CAPSULE

Microscopic examination of the mucilaginous deposit found
in cultures upon maple sap as described under "Cultural Char-
acters" revealed an envelope of almost transparent material and
about 1 micron in thickness covering the organism. In unstained
preparations, it was most readily observed by first placing the
organisms in perfect focus with the diaphragm slightly open, and
then reducing the light, when the colorless envelope became faintly
though distinctly visible, presenting a well defined periphery.
Organisms from the surface growth of carbohydrate agars showed
a similar capsulation when mounted without the use of water.
Organisms from carbohydrate agar condensation water or from
milk showed a similar phenomenon, but the envelope was less
clearly defined and scarcely more than .5 micron in thickness.
Stained preparations from maple sap and milk cultures were dis-
appointing. Sap preparations left to dry in the air formed a gela-
tinous mass about one-half the size of the original drop of
culture placed on the cover slip, and the volume of this material
could not be further reduced in drying with heat without charring
the preparation. Richard Muir's capsule stain was tried repeat-
edly upon these preparations, but washing in water after the
first mordant almost instantly dissolved the gelatinous mass and
left the cover slips without a film of organisms. This difficulty
was partially obviated by fixing in glacial acetic acid before the
mordant and substituting a 2'/< salt solution for water in the
subsequent washing. The stain, carbol fuchsin, was inactive

Micro-organisms oi? Maple Sap ls;i

upon the material even when used with heat for 20 I" 30 minuti
while the blue counter stain acted upon the organisms faintly.
Part of the bacilli <»n the films thus treated showed an unstained
envelope lighter than cither the body of the organism or the field

and from .5 to .75 micron in diameter. When Welch's capsule
stain was employed the organisms from sap cultures appeared
distinctly stained, surrounded by a transparent envelope .75 to 1
micron in thickness. Films from carbohydrate agar prepared by
Welch's method showed a colorless envelope upon practically
all organisms. Flagella preparations from 24 hour agar slants
stained by Loeffler's method with anilin gentian violet often ex-
hibited the capsule clearly stained. (Plate X. figure 1 ).

INVOLUTION FORMS

In a few cases oval refractive bodies suggestive of spores
were seen in preparations from cooked potato cultures several
weeks old. Transfers taken from such cultures were invariably
killed by heating for 10 minutes at 55° C. and attempts to stain
the bodies by the usual methods for spores gave negative results.
These bodies were deemed to be vacuoles.

STAINING REACTIONS

Preparations made from 24 hour old cultures upon nutrient
broth and nutrient agar were readily and deeply stained by cold
watery solutions of the anilin dyes, by Ehrlich's anilin water
gentian violet and by carbol fuchsin. Many of the rods stained
by carbol fuchsin exhibited plasmolysis. It is an interesting fact
that organisms from broth and agar cultures were deeply stained
by exposure for 10 seconds to cold carbol fuchsin. while this
stain used either cold or hot was totally ineffective with Richard
Muir's method of capsule staining applied to organisms from sap
cultures, as reported under the head "capsule." The organism
was decolorized by the method of Gram.

L84 Bulletin [67

Cultural Characters

METHODS

The culture media employ eel in this work were carefully pre-
pared following closely the directions given in Smith's "Bacteria
in Relation to Plant Diseases" and the publications of the Ameri-
can Public Health Association. Distilled water of a high degree
of purity was used unless otherwise stated. The formula em-
ployed for preparing- nutrient broth was 10 grams Witte's pep-
tone, 5 grams of Liebig's extract of beef, and one liter of distilled
water. Sodium chlorid was used only when so stated. All agar
media contained 1.5$ of agar flour. The reaction of media con-
taining nutrient broth was +10 Fuller's scale unless otherwise
noted. Titrations were made upon 5 cc. of medium diluted with
distilled water to 50 cc. The reaction was determined in hot
solution with N/20 sodium hydroxid against phenolphthalein.
All transfers except those for determining spore formation were
made from broth cultures 1 to 3 days old or from dilutions of
the same in water or, for some special purposes, in liquid culture
media. Transfers to fluid media were made with a 2 mm. plati-
num-iridium loop, and those to solid media with a straight needle.
Exceptions are noted in special cases as they occur.

Agar stroke. — Cultures developed good growth within 24
hours, which was moderate in amount, varying from a filiform
line to an cchinulately bordered, rather broad band. The eleva-
tion varied from raised to broadly umbillicate, with a surface at
first smooth but becoming faintly papillate and contoured in 4 or
5 days. Cultures 2 weeks or more old showed long villous or
sometimes fleecy outgrowths into the substratum. The growth
was translucent, accompanied by slight opalescence and a glisten-
ing luster. The culture was of slimy or butyrous consistency
without definite color, developing no discoloration of the medium.
The tubes gave off a mild yeasty odor.

Agar stab. — Young stab cultures in agar showed a growth
which at first was uniform along the line of puncture, later be-

Micro-organisms oi? Maple Sap lv-"'

coming better developed at the top, with a moderately spreading
surface growth. Development along- the line of puncture was at
first filiform and then echinulatc. Cultures 10 days old or more
were often beset with scattered tufts of villous or plumose out-
growths usually more pronounced in the upper part of the
medium.

Agar plates. — Colonies at 25 ° C. showed rapid growth. Those
at the surface were at first round, sometimes becoming slightly
irregular. In the early stages the surface was smooth, often
becoming contoured and papillate or developing moderate stria-
tions and concentric markings. These characters were not in-
frequently combined in the same colony. The elevation was
convex. The edge was at first entire or undulate, frequently
becoming lobate. The internal structure of surface colonies
showed all stages of variation from finely granular through
coarsely granular to grumose and, less frequently, reticulated.
The outer portion of many colonies was filamentous or curled.
Young cultures often showed an even outer ring well differen-
tiated from the body of the colony and consisting of motile chains
through which were thickly scattered granules. The appearance
suggested that which is often seen in very early stages of lique-
faction on gelatin plates, but there was no sign of liquefaction in
the agar. Young surface agar colonies 1 to 3 days old usually
showed active motility within the colony. Buried colonies first
appeared as brown lenticular bodies, often becoming irregular
with the interior deeply reticulated.

Carbohydrate agars. — Cultures were made in shaken agar
tubes containing 2% lactose, dextrose and sucrose, respectively.
Just previous to sterilization 1% of a solution of azolitmin (one
gram of azolitmin to 16 cc. of distilled water) was added to the
tubes. The sterilized melted agar was cooled to 400 C, inocu-
lated with a 2 mm. loop of a 24 hour old culture, thoroughly
mixed by shaking and incubated at 25 ° C.

In lactose litmus agar good growth developed and acid pro-
duction became evident during the first day. A heavy white film

1:86 Bulletin \(>y

of surface growth developed and during the third day gas pro-
duction began. This continued to increase in amount for a few-
days and then ceased. The reaction remained acid and at no time
was there evidence of reduction of the litmus.

In dextrose litmus agar the growth was more rapid than in
agar containing lactose. The acid production was more pro-
nounced and the gas formation observed was greater than was the
case with either lactose or sucrose. Signs of bleaching at the
surface appeared on the fifth day and progressed rapidly till the
entire contents of the tube assumed a pale yellowish hue. The
f< nination of a layer of liquid above the surface film of growth
was noted in most of the tubes under observation.

In sucrose litmus agar tubes the organism developed ven
much as in dextrose tubes except that the growth and the various
characters were slightly less pronounced, or at best developed a
little later.

Gelatin. — All gelatin media contained 10% of Nelson's
photographic gelatin No. I and was of such a consistency as to
remain firm at 250 C. Gelatin cultures were incubated at 200 C.

Gelatin stroke. — Gelatin slants showed a beaded growth along
the line of inoculation, frequently becoming filiform or echinu-
late, sometimes developing into a broad band with roundly dentate
margins and spreading beneath into an arborescent growth. After
a few days a fine filamentous outgrowth developed beneath the
surface under the stroke and extended several mm. into the body
of the gelatin. In some series this growth finally became arbo-
rescent while in others this character failed to develop to an ap-
preciable extent. Liquefaction of gelatin began on the eighteenth
day, and proceeded very slowly thereafter, becoming complete in
about three months.

Gelatin stab. — Stab cultures in gelatin showed best growth
at the top. The line of puncture was beaded, the beads soon
united to form a grannlose mass with a villous border developing
into long capillary fibers extending nearly to the walls of the
tube. These fibers eventually developed fine lateral branches,

Micro-organisms of Maple Sap 1v.

thus producing- a cloudy appearance. A characteristic surface
colony was formed. The medium remained unchanged until the
eighteenth to twentieth day when the first signs of liquefaction
were observed. The action was very feeble but persistent. Tubes
were completely liquefied in from 60 to 80 days. The cultures
30 days old exhibited infundibuliform or slightly napiform areas
of liquefaction extending from the surface down about 15 mm.
A heavy sediment deposited at the base of the liquefied portion
which was clear above and covered by a firm, dry layer which had
to be broken with a needle or by violent shaking in order to allow
the liquefied portion to run out when the tube was inverted.

Gelatin colonics. — Growth upon gelatin plates developed
rapidly at 200 C, producing colonies at first round and finally
becoming deeply lobed under favorable moisture conditions. The
elevation of growth was convex. During the early stages the
colonies presented an entire edge becoming undulate the second
• lay, and thereafter gradually becoming deeply lobate. The in-
ternal structure was at first finely granular becoming deeply
reticulate or alveolar and developing a great variety of markings
during the transition. Colonies one week old were strikingly
characteristic, having a dark alveolar center surrounded by an
intermediate lighter zone bearing- finely reticulate markings, and
verging into the outer zone consisting of deeply cut compact
lobes composed of conglomerate aggregates, as if they had been
formed by repeated expanding, bursting and reforming of an
enveloping pseudomembrane. Liquefaction was not observed
upon gelatin plates. (Plate IX).

Broth. — Cultures showed rapid growth and became semi-
opaque from clouding in 24 hours at 25 ° C. The clouding in-
creased for several days and was accompanied by the formation
of a flaky or somewhat membranous pellicle. The sediment at
first viscid became flaky in from 2 to 5 days. Cultures 6 to 8
weeks old were free from clouding with no pellicle but contained
a sediment which was viscid on agitation. The broth at this age

4vv Bulletin 167

took on a dark rich amber color and reacted distinctly alkaline
to litmus.

Potato blocks. — Cultures upon cooked potato blocks showed
a persistent growth which was moderate in amount, echinulate.
often spreading at the base ; convex with a glistening luster, con-
toured surface, and slimy to butyrous consistency. The tubes
gave off a characteristic odor suggestive of alcohol, and gas pro-
duction was noted. The medium was grayed.

Milk. — Tubes of fresh centrifuged milk inoculated with a 2
mm. loop of 24 to 48 hour old broth cultures held at 25 ° C.
showed no visible change for 4 to 6 days. Thereafter coagula-
tion occurred with gradual shrinking of the curd and extrusion
of whey, accompanied by gas formation. The same changes were
observed both at 20 and at 30° C, but the process was always
slower at the lower temperature and more rapid at the higher.
The coagulum was firm and leathery, becoming slightly yellowish
by reflected light. Tubes held under observation for 2 months
showed yellowish translucent spots which suggested slight pep-
tonization, but they were believed to result merely from shrink-
ing of the curd. (See production of proteolytic enzyms, pages

509)-

Acid production in milk. — This was determined in the fol-
lowing manner : Freshly drawn milk was centrifuged, then
further freed from fat by filtering through several thicknesses of
filter paper, carefully pipetted into tubes, 10 cc. in a tube, steril-
ized by discontinuous steaming on 3 consecutive days, and in-
oculated with a 2 mm. loop from young broth cultures. Titra-
tions were made at the end of 1,2, 4, 10, and 20 days, respectively.
For this purpose the content of the tube was added to 40 cc. of
distilled water, the mixture boiled for 1 minute and at once
titrated against phenolphthalein with N/20 sodium hydroxid. The
acid production was calculated by subtracting from the reaction
of the inoculated tube the average reaction of all check tubes in
the same scries and averaging the differences thus obtained.

Mil RO ORGANISMS OJ? MAI'LJJ SAP

489

TABLE 41. Arm PKODU< ikin i\ MILK

Series 1, 20

0 C.

Series 2,

20°

C.

Reaction in

Acid

Reaction in

Acid

Fuller's scale

production

Fuller's scale

production

Ag<' of

inoculated

in cc. N/1

inoculated

in cc. N/1

culture

tubes

control

per liter

tubes

control

per liter

1 day,

19.7

12.0

15.0

14.6

3.0

1 day,

19.2

7.3

18.2

2 days,

28.3

9.9

25.0

L3.3

10.7

2 days,

25.6

12.5

14.6

23.6

■1 days,

39.4

12.4

26.7

13.2

L3.7

4 days,

33.6

10.1

24.2

28.0

10 days,

46.6

13.0

36.7

14.0

25.8

10 days,

39.6

15.0

30.9

42.1

20 days,

49.1

11.4

lost

20 days,

41.4
of controls

12.9

33.0

lost

Average

12.20

13.6

Series 3, 25

0 C.

Series 4,

25°

C.

1 day,

28.8

17.8

27.5

1 day,

27.8

17.8

9.6

27.8

18.4

8.5

2 days,

32.3

20.0

34.0

2 days,

32.6

20.5

13.3

30.5

18.8

13.1

4 days,

32.0

19.5

37.8

4 days,

32.0

19.8

12.8

Q8 Q

t>o.o

20.0

18.9

10 days,

37.0

18.8

48.3

10 days,

37.0

19.0

17.8

46.3

19.6

28.1

20 days,

48.3

19.0

54.5

20 days,

46.8
of controls

19.4

28.4

52.0

19.2

34.1

Average

19.2

19.2

Series 5, 25

a C.

Series 6,

25°

C.

1 day,

18.7

10.2

20.0

13.7

8.0

1 day,

18.4

8.8

8.4

22.7

2 days,

27.1

9.1

28.0

12.5

13.7

2 days,

26.9

> • ■ .

16.8

26.0

4 days,

27.1

9.1

35.5

12.8

22.9

4 days,

27.1

8.5

16.9

37.0

10 days,

39.9

12.1

40.0

14.2

28.4

10 days,

38.3

....

28.9

45.5

20 days,

45.4

12.1

48.0

20 days,

48.3
of controls

11.7

36.7

48.6

13.3

35.0

Average

10.2

13.3

490

Bulletin 167

acid production — Continued

Series 7, 2.

3° C.

Series 8, 30° C.

Reaction in

Acid

Reaction in Acid

Fullei

's scale

production

Fuller'

s scale production

Age of

inoculated

in cc. N/1 inoculated

in cc. N/1

culture

tubes

control

per lite

r tubes

control per liter

1 day,

23.9

12. G

10.6

26.2

10.0 13.8

1 day,

21.2

10.0

25.3

....

2 days,

29.1

10.9

14.0

28.6

12.2 18.8

2 days,

22.9

12.3

32.9

12.8

4 days,

42.9

11.9

24.7

37.8

15.0 27.5

4 days,

30.4

12.7

41.1

10.6

10 days,

46.6

12.2

33.9

48.5

13.8 31.9

10 days,

45.0

. . • •

39.2

13.6

2(i days.

50.5

11.4

38.6

54.5

13.2 43.3

20 days,

of controls,

12.9

56.0

15.9

Average

11.93

11.94

Series

9, 30

0 C.

Age of

Reaction in

Fuller's

scale

Acid production

culture

inoculated tubes Controls

in cc. N/1 per liter

1 day,

24.7

13.5

11.8

1 day,

26.2

2 days.

33.5

13.0

21.1

2 days,

36.0

4 days,

42.4

11.0

27.1

4 days.

39.0

10 days,

44.0

14.2

30.7

10 days,

44.7

20 days.

57.8

14.3

43.9

20 days,

58.6

Average of controls,

13.8

TABLE 42. AVERAGE ACID PRODUCTION IN MILE IN CC. N 1 PER LITER

20° C.

25° C

1 day,

5.15

9.02

2 days,

12.65

14.18

4 days,

18.95

19.24

10 days.

28.:::,

27.42

20 days,

33.00

34.56

30° C.

12.80

19.95

27.30

31.30

43.60

Micro-organisms of M aim.k Sap P.* I

Litmus milk was prepared by adding- 0.10% of azolitmin 1"
fresh centrifuged milk and filtering through several thicknesses
m!" filter paper. Acid production became apparent within 24 hours
at 20, 25 and 300 C. followed by gas production, coagulation,
and extrusion of the whey, as reported under milk. Gradual hut
complete reduction of the litmus was observed beginning at the
top of the tube about the sixth day and becoming complete about
the sixteenth day. No further change was observed in tubes
held under observation for 3 months.

Starch jelly was prepared according to the directions given
by Smith. The nutrient material employed was the same author's
modification of Uschinsky solution. Good growth occurred upon
this within 24 hours. A broad band of raised growth, higher at
the border than at the center, with a papillate surface, an echinu-
late edge and a faint opalescence, resulted. Slight local lique-
faction of the medium was noted on the sixth day accompanied by
a change from opalescence to opaqueness with gas production in
the medium. On the tenth day and at intervals thereafter for six
weeks, tubes were examined by washing with distilled water.
Portions of the starch jelly had been rendered soluble so that
pockets were left in the surface. The wash- water and dissolved
material was passed through filter paper and tested for sugar
with Fehling's solution, with negative results. A portion of the
filtrate was distilled and the distillate tested with sodium hydroxid
and iodine as described under alcohol production. Iodoform was
produced, indicating the presence of alcohol, aldehyde, or ketone.
From the fact that Fehling's solution was not reduced the prob-
ability is strongly in favor of the supposition that the body was
an alcohol.

Cohn's solution proved an unfavorable medium for the culti-
vation of this organism. Cultures held under observation for 21
days failed to show growth.

Uschinsky solution. — Cultures developed a copious growth
appearing within 24 hours, accompanied by a pellicle formation
which after about 3 days sank and deposited as a membranous

4Q2 Bulletin 167

sediment. The medium was slightly viscid at the end of 2 weeks.

Fermi's solution inoculated with organisms developed a slight
growth within 24 hours, followed by the formation of a thin
pellicle which sank and formed a thin membranous sediment.
The growth was transient, the tubes becoming almost clear in
3 days.

Silicate jelly containing Fermi's solution showed a light
growth, becoming apparent the second or third day as a thin,
pearly white, surface development. The medium and growth
thereafter remained unchanged.

Sodium chlorid in bouillon. — Transfers were made to bouil-
lon ( + 10 Fuller's scale) containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
II, and 12%, respectively, of chemically pure sodium chlorid.
Growth promptly appeared in the four weaker solutions, but was
very slightly restricted in 5% sodium chlorid. It invariably ap-
peared upon the 6% and 7% solutions within 4 days and upon
the 8% and 9% ones in from 3 to 7 days. No growth occurred
in 10%, 11%, or 12% sodium chlorid bouillon within 21 days.
Transfers from these tubes into normal bouillon gave no growth.
In no case was development entirely inhibited by less than 10%
of sodium chlorid, but cultures in solutions containing from 5%
to 9% were not characteristic. Microscopic examination showed
organisms to be grouped together in chains in the 5% solution.
No signs of motility were observed in this or stronger concentra-
tions. In the broths containing in excess of 5% the growth was
restricted to clustered colonies gathered on the sides of the tubes.
In solutions up to 4% growth was apparently normal, the group-
ing and motility being characteristic.

Grozvth in bouillon over chloroform was unrestricted and
characteristic.

Maple sap sterilised by heating at 99 to ioo° C. on three
consecutive days and inoculated with a 2 mm. loop of the organ-
ism, developed a deep milky appearance within 24 hours. Growth
was accompanied by the formation of mucilaginous gum on the
walls of the flask in contact with the culture fluid. This layer

Micro-organisms of Maple Sap 493

increased in amount for one or two days, but began to diminish
on the fourth day and was entirely gone on the sixth daw Mean-
time the milky appearance of the sap remained unchanged. A
slimy consistency developed and the material sometimes became
stringy, but the pronounced ropy character described under
cultures in unsterilized sap did not appear.

Unsterilized sap was employed in a large number of the
inoculation experiments. Care was taken to procure this fresh
from the trees and as slightly contaminated as possible by bits of
dust, bark, and other extraneous matter, in order that the natural
inoculations should be minimized. In the first series a young
culture in beef broth was employed for the inoculations, about 5
cc. of culture being added to a liter of sap ; in other series vary-
ing small amounts of broth culture were employed, and in still
others pure cultures upon sterilized sap were used to produce the
inoculations. The cultural characters were invariably the same,
except that the heavier inoculations produced the results more
quickly. The sap took on a deep milky appearance but developed
no pellicle. A slime was deposited upon the walls of the con-
taining vessel but the gummy consistency of the deposit described
under sterilized sap was not observed. Moreover the liquid
developed a ropy character which was very pronounced after 24
to 48 hours and persisted for the two weeks that the cultures were
held under observation. The character of the culture produced
by inoculation with the organism after it had been cultivated in
the laboratory for one year was exactly similar to that observed
in the original material from which the organism was isolated.
The maximum reaction observed in unsterilized sap in which
this organism was cultivated for three days was h:7 Fuller's
scale. Before inoculation the reaction was +.3. Associated or
ganisms, when introduced in maple sap in such quantities as (<>
cause an overgrowth, produce a reaction of -f-.6 to -|-i.

Artificial maple sap was prepared from maple sirup as fol-
lows : 25 cc. of sirup, 475 cc. of water and 25 cc. of nutrient
bouillon were mixed, heated to boiling, filtered through filter

494 Bulletin 167

paper and sterilized in flowing steam on each of three consecutive
days. When inoculated this medium clouded promptly, becom-
ing milky. Ten day old cultures were slightly opalescent and
very milky, but neither pellicle nor sediment developed. The
reaction as shown by titration against phenolphthalein was -J-.81
Fuller's scale. Fermentation tubes filled with this medium
showed equally good growth in the open and closed arms, charac-
terized by a deep milky appearance and moderate gas production.
The material became viscid and sometimes a little stringy. From
5 to 15% of the closed arm was filled with gas consisting of
carbon dioxid and hydrogen.

Physical and Biochemical Features

Gas production in milk. — Fermentation tubes containing
milk inoculated with a 2 mm. loop of a young culture of the
organism and plated at 200 C. showed a small bubble of gas in
the closed arm at the end of two days ; otherwise the milk was
unchanged in appearance. Held at 25 ° C, the tubes of the same
age showed gas in 8% of the closed arm, while those held at 300
contained from 15 to 20(/o. On the third day at 200, 5% had
developed, at 25 °, 15 to 20%, and at 30 °, 40 to 45%. The ap-
pearance of the milk still remained unchanged. Cultures 10 days
old showed varying amounts of gas up to 100% of the closed
arm. The milk was coagulated and the firm coagulum usually
remained in the closed arm attached to one side of the tube.
About 70% of the gas produced was absorbed by sodium hy-
droxid and the remainder gave a slight explosion upon ignition
in air.

Carbohydrate broth. — Fermentation tube cultures of nutrient
bouillon containing dextrose, sucrose, lactose, maltose, glycerin,
and mannit were observed for gas production and acid produc-
tion. Titrations were made at the end of the first, second, and
fourth days, using 5 cc. of the culture in 45 cc. of boiling water
against phenolphthalein. Dextrose tubes showed rapid develop-
ment of the organism. At 200 the tubes became cloudy through-

Micro-organisms of Man.k Sap t95

out within 15 hours after inoculation, while at 25 and 300 ^;is
production was well under way. The growth in lactose and
sucrose was similar to that in dextrose but less pronounced.
Maltose was especially favorable for the development of the or-
ganism and abundant gas production was obtained upon this
sugar. Glycerin broth appeared a good medium for growth in
the open arm, but only restricted growth occurred in the closed
arm, and no gas was produced. Mannit gave good growth in
both the open and closed arms accompanied by gas production.
The tables showing acid production follow.

196 Bulletin 167

TABLE 13. ACID PKODUCTION 1 N CABBOHYDBATE B» ■ I 1 1

Reaction

in Fuller's

scale

Acid

Acid

Acid

Inocu-

production

Inocu-

production

Inocu-

production

Age of

lated

in cc. N/1

lated

in cc. N/1

lated

in cc. N/1

culture

tubes

per liter

tubes

per liter

tubes

per liter

Series

1-3. Dextrose

At

20° C.1

At

25° C.

At

30° C.

1 day,

14.0

4.0

17.0

7.0

20.0

10.0

1 day,

15.0

5.0

18.0

8.0

20.0

10.0

2 days,

21.2

11.2

23.0

13.0

22.5

12.5

2 days,

21.2

11.2

24.5

14.5

24.0

14.0

4 days,

19.5

9.5

21.5

11.5

15.3

5.3

4 days,

19.5

9.5

21.2

11.2

16.0

6.0

Series 4-6. Sucrose
At 20° C.2 At 25° C.

At 30° C.

day,

day.

days,

days.

days,

1 days,

1 1.5
14.5
16.2
17.2

17.5
18.5

5.0

16.5

7.0

19.5

10.0

5.0

17.0

7.5

19.5

10.0

6.7

20.0

10.5

19.5

10.0

7.7

19.0

9.5

10.3

10.8

8.0

16.5

7.0

16.8

7.3

9.0

16.5

7.0

17.7

8.2

Series 7-9. Lactose
At 20° C.3 At 25° C.

1 Average
: Average
r Average

of controls. 10.0.
of controls, 9.5.
of controls, 9.5.

At 30° C.

1 day,

12.0

2.5

L6.5

7.0

20.0

10.5

1 day

11.0

1.5

17.5

8.0

20.0

10.5

2 days,

L5.5

6.0

16.S

7.3

19.0

9.5

2 days,

15.5

6.0

16.7

7.2

18.0

8 5

4 days,

16.0

6.5

18.0

8.5

18.0

8.5

4 days.

17.0

7.5

19.0

9.5

18.5

9.0

Micro-organisms <u- Maim; Sap

I1.'

L'Able 43 — Continued

Reaction

in Fuller's

scale

Acid

Acid

Acid

Inocu-

production

1 undi-

production

Inocu-

product inn

Age of

lated

in cc. N/1

luted

in cc. N/1

lated

in cc. N/1

culture

tubes

per liter

tubes

per liter

tubes

per liter

Series

11-12.

Maltose

At

20° C

At

25° C.

At

30° C.

1 day,

14.2

2.8

l.s.::

6.9

17.5

t;.i

1 day,

L5.7

4.3

18.7

7.::

L6.5

5.1

2 days,

16.2

4.8

16.5

5.1

18.0

6.6

2 days,

1G.5

5.1

L6.5

5.1

17. t;

6.2

4 days,

19. G

8.2

18.9

7..")

18.2

6.8

4 days,

17.8

G.4

17.6

6.2

19.0

7.6

Series

13-ir..

Glycerin

At

20° C.3

At

25° C.

At

30° C.

1 day,

9.0

—0.6

8.2

—1.4

9.7

o.l

1 day,

9.3

—0.3

8.5

—1.1

10.5

0.9

2 days,

11.2

1.6

10.3

0.7

10.9

1.:;

2 days,

11.2

1.6

10.5

0.9

10.9

L.3

4 clays,

11.0

1.4

11.1

1.5

13.7

1.1

4 days,

11.7

2.1

11.4

1.8

13.7

l.i

Series 16-18. Mannit
At 20° C.6 At 25° C.

At 30° C.

1 day,

12.0

4.0

14.0

6.0

15.0

7.0

1 day,

14.0

6.0

12.8

4.8

L5.1

7.1

2 days,

14.7

6.7

16.1

8.1

17.8

9.8

2 days,

15.3

7.:;

15.7

7.7

L6.0

8.0

4 days,

16.4

6.4

16.8

8.8

L9.6

ii. i;

4 days,

17.0

9.0

17.9

9.9

20.2

L2.2

4 Average

Of (

contents, 11.4.

5 Average

of

controls,

9.6.

8 Average

of

controls,

8.0.

4!)S

Bulletin 167

The rate of gas production in the closed arms of the Smith
tubes was followed by measuring with the Frost card. The
amount of carbon dioxid formed was determined approximately
by absorbing it within the tube with sodium hydroxid, the volume
being noted before and after absorption. The residue was sub-
jected to the flame test for hydrogen. A positive reaction in-
variably resulted, although the explosion was frequently so faint
as to arouse suspicion that the material tested might be a mixture
of gases rather than pure hydrogen. The tabulated results fol-
low. The figures express the percents of the closed arm of
Smith tubes which were filled with gas.

TABLE 44. liAS PRODUCTION IN

CARBOHYDRATE

BROTHS

Age

Temperature

Dextrose

Lactose

•

Sucrose

f 20°

C.

trace

0

0

1 day,

-J 25°

C.

5%

trace

5%

[30°

c.

15%

8%

14%

[20°

c.

15%

:'.',

V,

2 days,

] 25°

c.

25%

20< ,

35%

[30°

c.

40%

25%

35%

[20°

c.

35%

25%

25%

4 days,

\ 25°
[30°

ABLE 45. P

c.

45%

40%

30%

c.

45%

IN',

50%

•j

PROPORTION

OF CARBON DIOXID

TO TOTAL

GAS

Series

1. Four

[lavs old

20°

C.

25°

C.

30°

C.

Percent

Percent

Percent

Percent Percent

Percent

of gas

CO.. to

of gas

CO .. to of gas

COsto

produced

total

produced

total

P

OdlK

■(•<]

total

I >extrose,

35

43

45

44

45

55

Lactose,

25

20

40

30

48

35

Sucrose,

25

40

30

45

50

50

Series 2.

Fourteen days old

Dextrose,

29

62

56

50

48

45

Ditto,

39

59

63

44

45

57

Ditto.

40

60

51

58

. .

Lactose,

37

75

48

58

36

72

Ditto,

40

72

35

63

41

61

Ditto,

40

70

55

60

:i

77

Sucrose,

26

50

49

53

48

. .

Ditto,

25

48

27

70

34

62

Ditto,

26

54

57

49

41

53

Micro-organisms of Maple Sap t99

Fermentation tubes of nutrient broth containing -''< of
mannit showed gas production in 24 hours at 25 ° C. but not at
200. At the end of two days 10% of gas had developed at 200
while at 300 25% had formed. Cultures six days old shown 1
a development of about 40% of gas, approximately one-half of
which was absorbed by sodium hydroxid. Evidences of fermen-
tation of maltose at 200 were pronounced in 15 hours, progress-
ing rapidly till 40 to 50% of gas was formed, which was similar
in composition to that produced upon other sugars. No gas was
produced upon glycerin broth.

Fermentation of potato starch. — Potato fermentation tubes
were prepared by placing cylinders of potato in the closed arm
and filling with distilled water. The tubes were sterilized by
steaming on each of three consecutive days, and inoculated with
a 2 mm. loop from young broth cultures. Gas production oc-
curred within 15 hours after inoculation in tubes held at 300.
At the end of three days tubes kept at 200 showed 25 to 35 per-
cent of the closed arm filled with gas. The gas wras tested with
sodium hydroxid and the flame test and found to contain carbon
dioxid and hydrogen.

Potato extract was prepared by boiling potatoes one-half
hour in distilled water and decanting the liquid upon a filter.
Fermentation tubes of the medium developed good growth. Gas
consisting of carbon dioxid and hydrogen was produced by the
second day. Tests for reducing sugars and for aldehydes made
on the third day negative results. These ingredients were sought
by the Fehling solution and ammoniacal silver nitrate methods,
respectively. Potato extract to which 4% of sucrose was added,
developed a remarkable growth. The fluid became milky and
slightly stringy and developed a yeasty odor very much like that
produced in sap.

Gas production in modified Uschinsky solution containing
carbohydrates. — To modified Uschinsky solution 2% of the fol-
lowing carbohydrates were added, maltose, dextrose, sucrose,
lactose and mannit. The media were placed in fermentation

500 Bulletin 167

tubes, sterilized by flowing steam on eacb of three consecutive
days, and inoculated in the usual way. At the end of 8 days gas
production in the closed arm was as follows: Maltose 22%,
dextrose 15%, sucrose 12/0, lactose $'-/> , and mannit 14%. The
composition of the gas was similar to that previously described.
Gas production in maple sap and in starch jelly containing modi-
fied Uschinsky solution has been mentioned under cultural char-
acters.

Ammonia production was determined in 100 cc. portions of
nutrient broth in 500 cc. flasks inoculated with 1 cc. portions of
young cultures and incubated at room temperatures, 20 to 23 ° C.
Determinations were made at the end of 5, 10 and 12 days. For
this purpose an excess of heavy magnesium oxid and 100 cc. of
distilled water were added. The flask was connected with a
Liebig condenser and distilled, the distillate being received in a
flask containing a measured quantity of N/20 hydrochloric acid.
The excess of acid was titrated against cochineal as an indicator
and the ammonia production calculated. An equal volume of the
same broth which had been kept under identical conditions was
analyzed for ammonia at the same time. The results are recorded
in the following table:

TABLE 46. AMMONIA PRODUCED IN NUTRIENT BROTH

cc. N/20HC1 cc. N/20NH

neutralized produced

Age Inoculated Control per 100 cc. broth

5 days, 11.80 — 2.00 = 9.80

10 days, 36.20 — 4.20 = 32.00

12 days, 33.15 — 4.75 = 28.40

12 days, 28.25 — 4.25 = 24.00

Nitrate reduction. — Nitrate broth was prepared according to
the following formula: One liter distilled water, 3 grams
Liebig' s extract of beef, 10 grams chemically pure potassium
nitrate. The reaction was adjusted to zero with sodium hy-
droxid. The organism made vigorous growth upon this medium
as exhibited by the prompt clouding and production of sediment.
There was a marked tendency towards clearing in the upper

Micro-organisms of Maple Sap 501

portion of the medium. Cultures were grown al j<>. 25, and 300
C. and tested for nitrites at the cud of 3, 5, 10 and 25 days with
prompt and positive reaction. The test was made with the fol-
lowing solutions :

(1) One gram of potato starch boiled in 100 cc. of distilled
water. (2) One-tenth gram of potassium iodid in 25 cc. of dis-
tilled water. (3) Two parts of chemically pure sulphuric acid
and one part water. To the tubes to be tested 1 cc. of number 1,
and 1 cc. of number 2, and, then, three drops of number 3 were
added, the tubes being agitated after each addition.

Indol production. — The tests for indol production were made
in Dunham's peptone solution and in sugar free bouillon. Both
media were well suited to the growth of the organism. The test
was made by adding 10 drops of chemically pure sulphuric acid
(two parts sulphuric acid to one part water). After allowing the
tubes to stand for 20 minutes to determine the absence of reduc-
ing bodies, 1 cc. of 0.02% sodium nitrite solution was added.
Cultures 10 days old on Dunham's peptone solution gave a pink
color in from 1 to 5 minutes after the addition of the nitrite.
Cultures grown at 200 responded somewhat more slowly and with
a fainter reaction than those which wrere held at higher tempera-
tures. As a check upon the work, tubes of the same medium
were inoculated with a strain of B. coli which produced indol
vigorously, and held at 300 C. Taking the color obtained from
these tubes as a standard, represented numerically by 10, cultures
of the organism held at 300 produced a color of 5 within one
minute. Cultures held at 20° produced a color of 1 after 15 or
20 minutes. A large number of cultures were tested in both
Dunham's solution and in sugar free bouillon and positive, though
sometimes faint, reaction was obtained in every case.

Production of phenol. — Tests for the production of phenol
were made upon 50 cc. portions of 10 day old broth cultures of
the organism. The material was transferred to a distilling flask,
5 cc. of concentrated hydrochloric acid added, and about 20 cc.
of distillate collected. This was divided into three portions.

502 Bulletin 167

One was treated with a few drops of Millon's reagent,1 another
with two or three drops of dilute solution of ferric chlorid, and
the third with a few drops of strong- bromine water.2 The results
were negative.

The reaction for phenol is sometimes obscured by the pres-
ence of traces of indol. To eliminate this source of error 200 cc.
cultures were distilled with 50 cc. of concentrated hydrochloric
acid. The 70 cc. of distillate obtained was rendered alkaline with
potassium hydroxid and again distilled. Indol should come over
in the distillate while phenol remains in the residue. The residue
was cooled, saturated with carbon dioxid and distilled. This
final distillate was tested as before for phenol with negative
results.

Hydrogen sulphid production. — This was determined in
bouillon tubes in the upper portion of which were suspended
strips of filter paper moistened with lead acetate solution. The
moisture was renewed with distilled water as was deemed neces-
sary. No signs of hydrogen sulphid appeared until the fourth
day, when traces of the black sulphid of lead appeared upon the
moist paper. The reaction became more pronounced a? the age
of the culture increased.

Toleration of acids. — Ordinary beef bouillon having a reac-
tion of o Fuller's scale was used in this work. Hydrochloric
and acetic acids were tested. Sufficient normal acid was added
to 100 cc. portions of bouillon to secure a reaction of +5, +10
and so on for every 5 degrees Fuller's scale up to +50. Good
growth resulted within 24 hours in tubes containing hydrochloric
acid up to and including +20. Growth was observed in tubes
having a reaction of -j-25 at the end of 2 days, and in 2 of the

1 Prepared by heating one part of mercury with two parts of nitric
acid, specific gravity 1.4, until the mercury was completely dissolved,
and then diluting the solution obtained with twice its volume of dis-
tilled water. Salkowski & Orndorff. Laboratory Manual of Physiologi-
cal and Pathological Chemistry, p. 251 (1904).

2 Chester, F. D. Manual of Determinative Bacteriology, p. ?..)
(1901). Salkowski & Orndorff, pp. 106 and 1G1.

Micro-organisms oi? Man.k Sap .r>".':

tubes having ;i reaction of +30 at the end of 4 days. No
further change occurred thereafter during the [9 days the tubes

were held under observation. Similar results were obtained upon
the second series, growth being obtained upon 3 out of 5 tub
having a reaction of +30. In the third series 4 out of 5 tubes
having a reaction of +3° showed growth. The organism was
less resistant to acetic than to hydrochloric acid. Feeble growth
occurred in 2 out of 5 of the tubes having a reaction of f-20
Fuller's scale, and in the weaker solutions of acetic acid the
growth was delayed and restricted.

Toleration- of sodium hydroxid. — For this work 100 cc. por-
tions of nutrient bouillon having a reaction of o Fuller's scale
were measured out and to each was added the theoretical amount
of N/i sodium hydroxid to produce reactions of — 5, — 10, etc.,
every 5 degrees of Fuller's scale up to — 45. The tubes were
inoculated as soon as possible after sterilization under pressure,
placed at room temperature, 20 to 240 C, and held under ob-
servation. Growth was observed at the end of 24 hours in
tubes calculated to have a reaction of — 5, and in one tube cal-
culated to have a reaction of — 10. At the end of 4 days growtli
was visible in all tubes calculated to have a reaction of — 15, and
in 2 out of 4 tubes calculated to have a reaction of — 20. At the
end of 7 days growth was observed in all tubes calculated for
— 20. At the end of 12 days growth was observed in all tubes
calculated to have a reaction of — 25. Thereafter no change oc-
curred during the 19 days the tubes were held under observation.
Transfers made from tubes which developed no growth after
19 days showed them to be sterile. Sterile control tubes of this
broth calculated to have a reaction of — 25, — 30 and — 35 were
titrated at the end of 20 days and found to have a reaction of
from — 19 to — 14. This change in reaction is of course to be
attributed to the formation of sodium carbonate from the carbon
dioxid of the atmosphere. The results however show that the
organism is killed when introduced to beef broth medium con-
taining sufficient sodium hydroxid to give a reaction of some-

504 l'.mj.KTiN 167

thing less than — 30 Fuller's scale, and that growth is at least
slightly restricted in broth containing sufficient sodium hydroxid
to give a reaction of — 10. The second series of tubes was pre-
pared in the same manner as those already described, inoculated,
and placed in sealed glass jars containing a few cc. of 2% sodium
hydroxid. At the end of 10 days the jars were opened and the
tubes examined for growth. Those calculated to have a reaction
of : — 5 all showed healthy development, but no growth was ob-
served in tubes having a more alkaline reaction. Titration of a
control tube of the — 5 broth showed that the reaction had not
fallen below — 4 Fuller's scale. Tubes calculated to have a reac-
tion of — 10 tested — 6.1 and — 6.8 and those calculated to be
— 15 tested — 10 and — II.

The third series was prepared in the same manner as the
second, but was incubated in Novy jars filled with air from
which the carbon dioxid had been removed by passing it through
wash bottles containing sodium hydroxid and calcium hydroxid
solutions respectively. The tubes were examined as well as
could be, without opening the jars, at the end of 7 days and
again at the end of 10 days. Growth was observed on both
occasions in tubes having reaction of — 5 but in no others. At
the end of 24 days the jars were opened and the tubes examined
critically for growth. All those calculated to have a reaction of
— 5 showed growth, but the stronger concentrations were in-
hibitory in all cases. Check tubes calculated to have a reaction of
— 5 were titrated and were found to react at from — 3 to — 5
Fuller's scale.

Optimum reaction. — As a basis for determining optimum
reaction, nutrient bouillon having a reaction of o Fuller's scale
was employed. Portions of 100 cc. each were treated with the
requisite amount of hydrochloric acid or sodium hydroxid to
give reactions at intervals of 5 points of Fuller's scale. Material
was tubed, sterilized, and inoculated with a 2 mm. loop of broth
cultures, and placed in the incubator at 250. The most vigorous
early growth occurred in tubes having reactions of +10 and

Micro-organisms of M mm k Sap 505

+ 15 Fuller's scale. The general appearance of the cultures
as regards turbidity, pellicle formation and rate of development
indicated that the optimum reaction is very close to -f-10 Fuller's
scale.

Temperature relations. — The optimum temperature was
determined in nutrient broth having reaction oi • i<> Fuller's
scale. Cultures were incubated at 20, 25, 30 and 370 C. The
best growth was obtained at 250 C. The maximum tempera-
ture was not much above 370 C. and only very feeble transient
growth was obtained at body heat. No attempt was made to
determine the minimum temperature, but growth was slow at
io° C.

Thermal death point determinations were made upon a
very large number of cultures. For this work thin walled test
tubes as uniform as possible and containing 10 cc. of nutrient
broth were used. Inoculations were made from 18 to 24 hour
old broth cultures. Great care, was taken to avoid slopping the
unheated inoculated broth upon the sides of the tubes above the
level of the medium. Within an hour after inoculation the
tubes were placed in a water bath so that the broth was well
below the surface of the bath, and heated for exactly 10 min-
utes. The temperature was kept constant within .1° C. 1>\ a
sensitive temperature regulator and a mechanical agitator.
After removal from the bath the tubes were cooled in air and
placed in the incubator at 25 ° C. The results of thermal death
point determinations were not absolutely uniform but in nearly
every case the death point was found to fall between 49 and
500 C. A few tubes heated at 500 showed growth, and in
some cases growth occurred after heating at a fraction of a
degree above 50. In a large majority of the cases the death
point fell between 49.5 and 500 C. In no case did death result
from heating for 10 minutes at less than 48.90 C.

Growth in carbon dio.vid atmosphere. — Tubes of nutrient
broth were heated to drive out the dissolved oxygen, co tied by
dipping in cold water, quickly inoculated, and placed with con-

506 Bulletin 167

trols in a Now jar, the cover of which was sealed with Darwin's
wax mixture. Carbon dioxid prepared in a Kipp generator
from boiled marble chips and chemically pure hydrochloric acid
was passed successively through wash bottles containing 10%
sodium carbonate, 10% potassium permanganate and boiled dis-
tilled water respectively, and then through the Novy jar. The
cotton plugs employed were as loose as was consistent with the
safety of the cultures and the jar was exhausted with the vacuum
apparatus several times to aid in removing all oxygen. After
carbon dioxid had passed through the jar two hours and a half
it was sealed and incubated at room temperature. After 20
days the jar was opened and the tubes examined for growth
with negative results. Within 24 hours after air was admitted
to the culture all except the controls developed characteristic
growth.

A second series of cultures was prepared in the same man-
ner as the first except that a 2.' < dextrose nutrient broth was
employed. Five days after inoculation fermentation became ap-
parent in several tubes, as could be seen from the gas collecting
in bubbles at the surface. After two weeks all inoculated tul>es
showed fair growth. Pellicle and ring formation were absent.
The growth was characterized by a tendency to granular forma-
tion throughout the medium and the evolution of appreciable
amounts of gas on agitation. When air was admitted to the
cultures moderate clouding promptly developed and the usual
aerobic characters appeared.

Desiccation. — For desiccation tests small portions of 24
hour old broth cultures were transferred to sterile cover slips
in sterile Petri dishes. Only a very small amount of material
w as transferred and this was spread in a thin film on the cover
slip. The preparations were allowed to dry at room tempera-
ture, 20 to 240. Slips were transferred to tubes of nutrient
broth with sterile forceps as soon as dry. and at the end of 3
hours. 17 hours. 24 hours. 4T hours, 50 hours, 6 days, 9 days, and
20 days. In no case was the organism killed by less than

MiCRO-OFGANiSMS 0* M ah.k S.\r ."'"7

6 davs drying. In one of the 5 tests made no growth was ob
tained in a tube inoculated with a cover slip dried for <> days.
Growth occurred 2 days after inoculation with cover slips dried
for i) days. No growth occurred in tubes inoculated with covel
slips dried for 20 days. Tubes were held under observation for
10 days after the cover slips were introduced.

Insolation. — Agar plates were sown with such dilutions as
to produce from 100 to 200 colonies, and allowed to stand until
thoroughly hardened. One-half of the plate was covered with
an opaque screen, and the cultures exposed on ice to the influence
of direct sunlight for 15 minutes. The exposure was made in
the middle of the day on August 28. After exi><>sure the plates
were returned to the incubator and allowed to remain at a
temperature of 250 for 3 days. The colonies were then counted
upon exposed and unexposed portions of the plate, with re-
sults as follows:

Exposed side

81

colonies

65

tt

58

a

16

n

20

a

5

a

13

tt

Total.

258

a

Difference,

226

tt

Unexposed side
117 colonies
85 "
80 "
27 "
50 "
85 "
40 "

484 "
Calculated percent killed, 46.7

Production of alcohol. — Four different media were em
ployed in the determination of alcohol production, viz.: 2'',
dextrose bouillon, 2% sucrose bouillon, potato broth, and maple
sap. Inoculation was made in liter flasks in 500 cc. portions of
media to which 10 gr. of calcium carbonate had been added im-
mediately before sterilization. The cultures were held at room
temperature, 20 to 240 C. After 10 days incubation the undis-
solved calcium carbonate was removed by filtration, the cultures
made slisrhtlv acid with hvdrochloric acid and then distinctly
alkaline with sodium carbonate, the precipitated calcium car-

508 Bulletin 167

1 M.nate removed, the filtrate distilled, and about 75 cc. of the
distillate collected. A portion of the distillate was tested for
alcohol and allied bodies by adding 5 to 6 drops of a 10',
S( dution of potassium hydroxid, bringing the solution to a
temperature of 500 C, and, then, adding a saturated solution
of iodin in potassium iodid drop by drop until the liquid had
a permanent brown color. After thorough agitation, the potas-
sium hydroxid solution was added drop by drop until the color
disappeared. In every instance a copious formation of iodoform
resulted. None of the controls gave the precipitate. This re-
action is not peculiar to alcohol, but is produced also by ace-
tone, aldehyde, isopropyl alcohol, propylic and butylic alcohols
and aldehydes, various ethers, meconic, laevulic. and lactic acids.
turpentine, sugar, etc. On the other hand it is not given by
methyl or amyl alcohol, chloroform, chloral, glycerin or ether;
nor by acetic, formic or oxalic acids.1 In general substances
containing the group CH,C linked to oxygen answer to the
iodoform test.2

The iodoform producing bodies likely to be present in
this distillate were alcohol or bodies belonging to the aldehyde
or ketone groups. In order to determine if possible whether
the body present was an alcohol, aldehyde or ketone, or whether
all three were present, portions of the distillate were subjected
to the following tests :

1. A few drops of 10 percent sodium hydroxid were added
to a few cc. No distinctive reaction occurred, but upon stand-
ing a small amount of slimy or slightly resinous precipitate
collected at the bottom of the tube. This was present, however.
in only minute amounts.

2. A solution of Merck's phenol dissolved in excess of
sulphuric acid when treated with a few cc. of the solution gave
no characteristic scarlet color.

'Allen. Commercial Organic Analysis. 1, p. 91 (1903).
2Holleman, A. F. Textbook of Organic Chemistry, p. 173 (1903).

M [CRO ORGANISMS OF M \ru; SAP 509

3. Hot Fehling's solution in excess was reduced by a por
tion of the distillate, giving characteristic copper precipitate

4. A saturated cooled solution of acid sodium sulphite
treated with a portion of the distillate gave no reaction.

5. The color of a solution of fuchsin, mixed with just
sufficient sodium sulphite almost to decolorize it, was not re-
stored by the addition of 5 cc. of the distillate, although in
some cases there appeared to be a slight suggestion of return-
ing color.

6. A 10% solution of silver nitrate was treated with an
equal quantity of to'-' solution of potassium hydroxid ami am-
monia was added until the precipitate just dissolved. A 1
lion of the distillate added to this reagent gave a metallic mirror
after standing for half an hour, or within one or two minutes
when the tube containing the mixture was gently heated over
a flame.

The indications were that while aldehyde or ketone or
both might be present in small quantities, the greater part of
the iodoform must have been produced from alcohol. As a
further check upon this conclusion 50 cc. of the distillate produc-
ing- iodoform were added to an excess of hot Fehling's solution
and the containing flask immediately connected with a condenser
and distilled. About 20 cc. of the distillate were collected. An
examination of the residue in the Fehling's solution showed a
reduction of copper. The distillate gave a copious precipita-
tion of iodoform, but failed to react with ammoniacal silver
nitrate or with decolorized fuchsin.

Production of proteolytic ensyms. — The slow liquefaction
of gelatin in old cultures and the suggestion of partial digestion
observed in old milk cultures indicates that this organism pro-
duces proteolytic enzyms only when forced to do so and then
only in verv small quantities. In order to determine this point
fresh centrifuged milk was passed through several thicknes
of fine meshed filter paper, and finally freed from the last traces
of fat, and at the same time rendered sterile, by passing it

510 Bulletin 167

through a Pasteur filter. Portions of 50 cc. each were removed
to sterile flasks by means of sterile pipettes and inoculated with
the organism. The cultures were held at 25 ° C. and tested
for proteolytic action at intervals from 10 to 44 days, always
with negative results. The test was made by adding- to the
culture in the flask 75 grams of ammonium sulphate, heating in a
water bath at 6o° C. for 30 minutes to precipitate all proteids
except peptones and propeptones, filtering, rendering the filtrate
strongly alkaline with potassium hydroxid and testing with a
few drops of a 1% solution of copper sulphate.1

Production of diastolic ferments. — A thin starch paste to
which was added 2' ", of sugar free thymol was prepared and
added to equal parts of 10 day old broth cultures. After in-
cubation at 250 for 6 to 8 hours the solution was filtered and
tested for reducing sug"ar with Fchling's solution. The results
were negative.

Production of invertase. — A solution containing 2rr of cane
sugar and 2% of phenol was prepared for the purposes of
tin's test. Kqual quantities of 10 day old broth cultures of the
organism and this solution were thoroughly mixed and left in
the incubator over night. The unclarified solution when tested
with Fehling's solution gave a pronounced change of color with
a variable amount of copper precipitate, but the reaction was
so far from characteristic that a check upon the work was
desired. It is customary in analysis of solutions for invert
sugars to clarify with some substance, such as neutral lead ace-
tate or copper sulphate, before proceeding with the analysis.
In the use of lead acetate it is important to avoid a large ex-
cess and it is necessary to remove the slight excess used by pre-
cipitation with calcium oxalate before proceeding with the Fehling
test. In order to determine the influence of clarification of
bacterial cultures before running the Fehling test the following
solutions were prepared :

1 Salkowski and Orndorff, loc. cit., pp. 15 and 40.

Micro-organisms of Maple Sap 7,11

i. A solution containing- 2^ of phenol, 2% of sucrose, and
._•' , of dextrose.

2. A solution containing 2'/< of phenol, 2% of sucrose, and
.2% of dextrose, together with an equal volume of sterile bouillon.

3. A solution containing- 2% of phenol, 2% of sucrose, .2' -
of dextrose and an equal volume of a io day old broth culture of
the organism.

4. A solution containing 2% of phenol, and 2r/< of sucrost

5. A solution containing 2% of phenol, and _>' , of sucrose,
together with an equal volume of sterile bouillon.

6. A solution containing 2'.', of phenol. _>', of sucrose.
together with an equal volume of a 10 day old broth culture of the
organism.

7. A solution containing 2% of phenol.

8. A solution containing 2% of phenol, together with an
equal volume of sterile bouillon.

9. A solution containing 2% phenol, together with an equal
volume of a 10 day old broth culture of the organism.

10. A solution containing sterile bouillon.

11. A solution containing- a 10 day old broth culture of the
organism.

Three 10 cc. portions A. B, and C of each solution were taken.
To the solutions A, 1 cc. of a strong solution of lead acetate wa^
added. They were filtered, the excess of lead acetate precipitated
with potassium oxalate crystals, care being taken to avoid an
excess of potassium oxalate. The lead oxalate was filtered off
and the filtrate reserved for treatment with Fehling's solution.
Portions B were treated with 1 cc. of copper sulphate solution
used in connection with the Fehling's solution. Portions C were
given no preliminary treatment before using the Fehling test.
The precipitation was carried out in Erlenmeyer flasks. By
means of a pipette 10 cc. of the copper solution was placed in the
flask, then 10 cc. of the tartrate, care being taken to avoid gather-
ing undissolved precipitate on the walls. The two were mixed
and heated t<» boiling. A 10 cc. portion of the solution to he

512 Bulletin 167

tested was added to the mixture which was again brought to a
boil and maintained at gentle ebullition for 2 minutes. It was
then removed from the hot plate and transferred to a porcelain
evaporating dish and left to settle for a few minutes. The liquid
was decanted and the bottom of the dish examined for precipitated
copper. The following table shows the results obtained. The
presence of precipitated copper is indicated by the sign -)-, and
its absence by the sign — . Where only a very minute precipitate
was obtained it is indicated by the word "trace."

A

B

C

Character of solution

Prelim'y

Lead

Copper

treatment

acetate

sulphate

1

+

+

+

phenol, sucrose, dextrose.

2

+

+

+

phenol, sucrose, dextrose,

bouillon.

3

+

+

+

phenol, sucrose, dextrose,

culture.

4

—

trace

trace

phenol, sucrose.

5

—

—

—

phenol, sucrose, bouillon.

G

trace

trace

+

phenol, sucrose, culture.

7

1 race

1 race

—

phenol.

S

—

—

— r

phenol, bouillon.

!)

—

—

—

phenol, culture.

10

—

—

—

bouillon.

11

—

—

—

culture.

The reaction in scries A and B was in general characteristic.
In series C, numbers 1 and 4 were characteristic, number 2 was
nearly so, while numbers 3 and 6 gave a muddy solution with an
uncharacteristic color and, in addition to the copper oxid, the pre-
cipitate contained a brown, light weight, flocculent material.

Cultures upon potato slants 4 days old were washed with
distilled water, the washings were filtered and tested for reducing
sugars with Fehling's solution with negative results.

Effect of germicides.— The germicidal effect of formaldehyde
and of phenol were tested in broth cultures of the organism by
adding definite known quantities of these germicides to tubes of
sterile medium and then inoculating with a 2 mm. loop of young
broth culture.

Formaldehyde. — As a basis r\.r this work a guaranteed 40
formaldehyde was employed. < me cc. of the solution was added

J0

m

Plate XI. — Ps. fluorescens var. non-liquifaciens, strain CXI.. Flagella
preparations from 24-hour agar slant, (Loeffler's stain). (See
page 555. ) X 1500.

Plate XII. — Ps. fluoresce)!*. Types of gelatin colonies. Figures 1 and
2. Strain LIII (two day colony). Figures 3 and 4. Strains LVI
;in<] r. it^o <i -i a- oninnioot Fieui'ae R. P ppri 7. Strains XXXIII
(two day colony) XXXVI and XXXIII (one day colonies). (See
pages 5i;7-5(is. ) X 75.

Micro-organisms of Maple Sap 518

to flasks containing 49. 99, 149 and [99 cc. respectively of dis-
tilled water. From each of these flasks portions of 1 cc. were with-
drawn with sterile pipettes and placed in test tubes containing
10 cc. of broth, thus making dilutions of 1-550, 1-1100, 1-1650
and 1-2200 respectively. Eight tubes of each dilution were pre-
pared, 5 tubes were inoculated with a 2 mm. loop of a 24 hour old
broth culture, and 3 were retained as controls. Incubation was at
250 C. No growth had developed in any of the tubes at the end
of 2 days. On the fourth day growth was observed in one of the
five tubes containing- 1 part of formalin to 2200 of water. Thir-
teen days after inoculation transfers were made into sterile broth.
The transfers from the tube showing growth developed a charac-
teristic appearance but those from the other tubes remained un-
changed. In the second series 1 cc. of 40/Y formaldehyde was
added to flasks containing' 199, 249 and 299 cc. respectively of
distilled water. One cc. portions were transferred with sterile
pipettes to tubes containing- 10 cc. Of broth, thus making dilutions
of approximately 1 part of formalin in 2200, 2750, and 3300 parts
respectively. As in the previous instance 5 tubes were inoculated
and 3 retained as checks. \Vithin24 hours growth appeared in 3
of the 5 tubes containing- 1 part of formalin to 3300. The second
day growth was evident in all of the tubes containing 1 part of
formalin to 3300 and in one of the tubes containing 1 part to
2750. The cultures f> days old showed growth in all tubes of the
weakest dilution, in 3 of the 5 tubes containing t part formalin to
2750 of the medium, and a very slight growth in one of the tubes
containing- 1 to 2200. ( )n the eleventh day the tubes had all
cleared except one containing formalin in the proportion of 1 to
3300. This tube showed strong clouding.

Phenol. — The phenol employed in this work was Merck's
phenol, U. S. P. VII. This was melted by placing the container
in warm water. To a portion was added 1 1 ' < of water h\
weight, thus obtaining a solution which remained liquid at ordin-
ary temperatures. One cc. of this liquid phenol was added to
9, 49, 99, 149, and 199 cc. respectively of distilled water. One

514

BUIXKTIN l67

cc. portions of these various dilutions were added with sterile
pipettes to tubes containing 9 cc. of sterile broth, thus making
dilutions of 1-100, 1-500, 1-1000, 1-1500, and 1-2000 of liquid
phenol in broth. As in the case of the formalin 5 tubes were
inoculated and 3 retained as checks. Growth was observed at
the end of a day in all tubes containing 1 part liquid phenol to
2000 parts of broth, and in those containing 1 part to 1500, while
four of the five tubes containing 1 part of liquid phenol to 1000
parts of broth, showed slight clouding. Good growth eventually de-
veloped in all tubes containing 1 part to 1000. No further change
was observed in these tubes during the 2 weeks they were held
under observation.

Nitrogen requirements. — The water used in the study of the
nitrogen requirements of the organism was re-distilled with potas-
sium permanganate, after which a third distillation employing
sulphuric acid was carried out. Only chemicals of known purity
were employed, except in the case of glycerin which, while of a
high grade of purity, was not free from traces of nitrogen.

The following formulas were used :

Sodium chlorid,
0 grams.

Calcium chlorid.
.10 gram.

Magnesium sulphate,
.35 gram.

Di-potassium phos-
phate, 2.2 grams.

Glycerin (Merck's blue
label), 35 grams.

Water, 1000 cc.

Sodium chlorid,
6 grams.

Calcium chlorid,
.10 gram.

Magnesium sulphate.
.35 gram.

Di-potassium phos-
phate, 2.2 grams.

Mannit, 35 grams.

Water, 1000 cc.

C.

Sodium chlorid,
G grams.

Calcium chlorid,
.10 gram.

Magnesium sulphate,
.35 gram.

Di-potassium phos-
phate, 2.2 grams.

Dextrose, 35 grams.

Water, 1000 cc.

Each of these solutions, A., B., and C., were divided into five
portions, one of which in each case was reserved as a control.
To the other four were added 1% of asparagin, urea, ammonium
chlorid, and potassium nitrate respectively. The media were
tubed and sterilized in the autoclave fur 15 minutes under 5
pounds of steam.

M uk iG vnisms "i .M.\i'i,i;' Sap 515

Series A. Glycerin. — As would be expected, growth devel-
oped in the control tubes of series A where glycerin was employed
as a source of energy, since they were not nitrogen free. Growth
became apparent the third day, but was slight and transient. The
tubes containing- potassium nitrate appeared exactly like the con-
trols. The asparagin tubes developed growth in six days which
increased, becoming- very pronounced in two weeks. At no
time did development occur in tubes containing urea or ammonium
chloric!.

Series B. Mannit. — No growth occurred in the controls or
in tubes containing potassium nitrate. Very feeble growth was
observed with ammonium chlorid and with urea. The asparagin
tubes showed growth the sixth day which became very pro
nounced. A dee]) milky color appeared, and a thick, tough, pel
liclc formation and heavy sediment developed.

Series C. Dextrose. — The controls gave no growth. Potas-
sium nitrate, ammonium chlorid and urea all developed feeble
growth, which was less transient in the case of ammonium chlorid
than with potassium nitrate. Asparagin again gave a luxuriant
growth after a period of delay. The development upon asparagin
was better when dextrose was used than when either glycerin or
mannit was employed. Moreover the organism seemed to be
able to make slight development upon potassium nitrate in as
ciation with dextrose but not when mannit was substituted, and
apparently not when glycerin was substituted. Urea and ammo
nium chlorid supported feeble growth with mannit and dextrose
but rtot with urea. The results are summarized in the following
table in which the sign — denotes no growth, + feeble growth.
"J: abundant growth after delay, and ? transient growth probably
attributable to nitrogen derived from the glycerin.

TABLE 47. GEOWTH OX NITBOGENOl S MEDIA

A.

Glycer

in

B.

Mannit

('.

Dextrose

Control,

9

—

—

Asparagin,

•;-

t

t

Urea,

—

+

+

Ammonium chlorid,

—

+

+

Potassium nitrate,

1

—

+

516 Bulletin 167

NAME

A careful comparison of the cultural and biochemical char-
acters of this organism with those of bacteria previously de-
scribed would seem to justify its recognition as a new species.
The name Bacillus aceris (new species) is therefore suggested
for it.

B. BRIEF DESCRIPTION OF THE PINK COCCI OF

MAPLE SAP1

Pink colonies were frequently found upon the plates poured
from sour sap, particularly late in the season. Some of these
were produced by yeasts or yeast-like organisms while others were
composed of micrococci. Four strains were selected for further
study from the cultures of pink micrococci isolated. These were
CXXVI, CIV, XL'IX. and CVI. A brief description of the or-
ganisms is given here for the purpose of record. All appear to
belong to the type of Micrococcus roscus. They resemble each
other closely and will be described as though identical, any varia-
tions in behavior from the general type being noted as they occur.

Morphology

Vegetative cells. — The organisms art.' coccoid in form, occur-
ring singly, in twos, fours, or irregular packets. Division is in
two planes. The average diameter as determined on agar hang-
ing1 block cultures was 1.2 microns. The smallest organisms ot>-
served measured .8 micron and the largest 2. microns. Cultures
in nutrient broth had a tendency to be somewhat smaller and to
occur singly or in twos.

Spores. — Evidences of spore formation were absent.

Flagella.— -The organisms were non-motile and no indica-
tions of flaij'ella were observed.

'The matter appearing on pages 516-521 is briefed from a thesis
entitled "The Pink Micro-Organisms of Maple Sap." presented by N. R.
Smith, B. S., of the class of 1911, in partial fulfillment of the require-
ments for the baccalaureate degree in the College of Agriculture of the
University of Vermont.

Micro-organisms of Maple Sap 517

Capsule. — There was a tendency to develop a slimy sediment
in liquid cultures, but microscopic examination and staining
methods failed to demonstrate a capsule.

Zooglea. — Xo evidence of zooglea masses was observed.

Involution forms. — Involution forms were not observed.

Staining reactions. — The organisms stained readily in 1-10
cold watery fuchsin, gentian violet, Loeffler's alkaline methylene
blue, and carbol fuchsin. The method of Gram gave negative or
sometimes doubtful results.

Cultural Characters

Agar stroke. — Growth was moderate becoming visible two
days after inoculation. It was raised, regular, slimy, and tinged
with pink. Iridescence developed after 10 days, the growth hav-
ing spread to a width of 4 to 5 mm.

Agar stab. — Slight growth became visible after 24 hours. The
second day a characteristic surface colony appeared. The growth
in old cultures (6 to 8 weeks) became villous along the line of
puncture. Colonies were first gray, and then became pink. When
7 days old they were from 2 to 4 mm. in diameter, raised, shiny,
and pigmented. The edge was regular, thin and transparent, and
the center opaque.

Two percent sucrose litmus agar. — Growth was confined to
the surface, and characterized by the production of pink pigment.
Acid was produced but the litmus was not reduced.

Two percent dextrose litmus agar. — Acid production became
apparent the second day. Reduction of the litmus began after
one week and progressed slowly until complete about the fourth
week.

Two percent lactose litmus agar. — Growth was slight, acid
formation and litmus reduction 1x)th being absent.

Five percent glycerin litmus agar. — The growth was slow
and acid production absent. Feeble litmus reduction was noted
on the fifth day. and thereafter progressed slowly, becoming com-
plete after two weeks.

518 I'.PI.I.KTIN 167

Potato. — Organisms failed to develop visible growth upon
cooked potato slants.

Gelatin stab. — Growth was slow, moderate, at first beaded
almig the line of puncture, becoming filiform above. Slight lique-
faction began on the twenty-fourth day and continued slowly,
becoming' stratiform in six weeks. The liquefied gelatin became
pinkish, contained a compact pink sediment and was distinctly
viscid.

Gelatin plate.— Colonies appeared the third day, and attained
a diameter of 2 mm. in 10 days. They were convex with round
entire edges. The pigment upon gelatin was similar to that upon
agar except that it was usually of a darker hue.

Broth. — First evidences of growth consisted of a slight, com-
pact, pinkish sediment appearing the second or third day, while
clouding appeared in one wreek. The sediment was viscid on
agitation.

Milk. — The first evidence of growth consisted of a depositr m
of pink sediment, becoming apparent within the first 10 days. In
3 weeks the medium was colored light pink while a watery ap-
pearance developed the fourth week. This appeared to be as-- >
ciated with feeble digestive action, although complete peptoniza-
tion did not occur. Coagulation did not take place.

Litmus milk. — The organisms studied developed considerable
variation upon this medium. CXXVI showed acid formation
after 14 days, with coagulation 2 or 3 days later, promptly fol-
lowed by the reduction of the litmus in the lower part of the tube.
Partial digestion of the curd occurred. The first evidences of
peptonization appeared during the third week and progressed
slowly until about the ninth week, when about half of the total
curd had become dissolved. CIV exhibited no change in the
medium until after 6 weeks, when it turned alkaline and curdled.
Digestion did not occur. XLIX became alkaline the third week
and developed a soft curd 3 or 4 days later. Litmus reduction
occurred after 4 weeks. Partial liquefaction of the coagulum was
observed in about half of the cultures vi this strain, but did not

Micro-organisms os M\m Sap 519

occur in the remainder. CVI curdled milk after 5 weeks with
reduction of litmus. The color was restored with au alkaline re-
action in the upper half of the tube after 8 weeks. No digestion
of casein was observed.

The variations upon litmus milk were so great that the work
was repeated several times and the purity of the cultures carefully
verified, but without change in the character of the reaction.

Cohn's solution. — A slow growth was noted which was char-
acterized by the development of sediment which usually was
granular and flaky, but sometimes slightly viscid.

Uschinsky solution. — The growth was slow, at first moderate
in amount, becoming abundant after 5 weeks, with copious sedi-
ment. A portion of the culture immediately above the sediment
exhibited clouding.

Growth in bouillon over chloroform. — There was no growth
111 bouillon tubes to which 2 or 3 drops of chloroform were added.

Physical and Biochemical Features

Acid production. — Nutrient broths to which were added 2' ,
of various sugars and 5^? of glycerin respectively were tested for
gas formation and acid production in fermentation tubes. In no
case was there sufficient gas production to become apparent with
the Smith tubes. Ten cc. portions were titrated in the usual way
at the end of I, 2, 4, 10, and 28 days. The reaction of duplicate
checks was subtracted from the average reaction of inoculated

luplicates to determine the average acid or alkali production.

The results expressed in percenl normal are shown in the follow-
ing- table.

f 1 *

520

I U I.I.I-TJX 167

TABLE 48. ACID PRODUCTION OX CARBOHYDRATE BROTHS

Dextrose

1 day

2 days

4 days

10 days

28 days

CXXVI,

—.13

—.08

—.05

.11

.45

CIV,

—.07

—.03

—.03

.22

.38

XLIX,

—.10

—.03

.07

.10

.40

CVI,

.10

.09

.00

.08

.39

Sucrose

CXXVI,

.06

.12

.03

.14

.50

CIV,

.08

.08

.03

.32

.02

XLIX,

.02

.05

.06

.07

.30

CVI,

.05

.10

.04

.11

.68

Lactose

CXXVI,

—.09

—.05

—.11

—.08

.60

CIV,

—.04

—.08

.06

—.10

.04

XLIX.

— .00

—.10

.01

—.08

.02

CVI,

—.03

—.04

—.08

—.08

.42

Glycerin

10 days

20 days

CXXVI,

—.05

.10

CIV,

.05

.00

XLIX,

.00

—.05

CVI,

—.24

.25

Nitrate reduction. — Cultures grown in nitrate broth were
tested for nitrites at the end of 5, to and 15 days, with uniformly
positive reaction. Other cultures were tested with Nessler's re-
agent for ammonia with negative results.

Hydrogen sulphid. — Cultures on nutrient broth were tested
for hydrogen sulphid production by means of moist filter paper,
bearing lead acetate, suspended from the plug. No evidences of
this gas were observed.

Indol production. — Cultures on Dunham's solution and on
peptone water were tested for indol production with negative
results.

Temperature relations. — The optimum temperature was from
20 to 250 C. The maximum was slightly above 400 C. Growth
occurred at io° but not at 40 C.

Thermal death point. — Thermal death point determinations
were made by exposing young transfers in thin wall test tubes at
various temperatures, in a constant water bath for ten minutes

Micro-organisms of Maple Sap 523

XLIX failed to grow in tubes exposed at 44 C. or alx>ve, Cl\ al
460 C, and CXXIV and CVI at 480 C.

Desiccation, — Sterile cover glasses were plaeed in Petri dishes
and inoculated, each with a loop of media from a 12 day old broth
culture, and allowed to dry for intervals varying from the first
instant when the cover glasses were free from the film of moisture
up to 30 days. Their viability was tested by transferring the
cover slips to tubes of broth by means of sterile forceps. The
cultures were held for two months but in every instance failed to
show growth.

Insolation. — Thinly sown agar plates were exposed on snow
to direct sunlight for 30 minutes with one-half covered. After a
period of incubation the colonies which developed on the exposed
and unexposed portions of the plates were counted. There was
practically no difference in the number of colonies developing.

Pigment formation. — Pigment formation occurred only in
the presence of oxygen and at ordinary temperatures. It failed
entirely at 37° C. Upon solid media such as agar and gelatin each
coccus had its peculiar tinge of pink. An attempt was made to
refer these colors to the chart given by Winslow,1 as follows:

CXXVI

=

Orange red

III & IV.

CIV

—

Orange red

IV.

XLIX

m

Orange yellow

VI.

CVI

z^^

Orange red

Ill & IV.

The pigment was insoluble in water, dilute sulphuric acid, xylol,
cold or hot alcohol, and chloroform. In dilute nitric acid the
growth was disintegrated and the pigment dissolved slightly.

C. THE GREEN FLUORESCENT BACTERIA
OCCURRING IN MAPLE SAP

Introduction

Bacteriological literature contains descriptions of more than
50 species of bacteria which are capable of producing a green
fluorescent pigment. Such bacteria were originally assigned to

1 Winslow. Systematic Relationships of the Coccaeea? (1908).

522 Bulletin 167

various widely separated groups, the early writers recognizing
the ability to form the fluorescing pigment as a valuable diagnostic

character, but apparently failing to consider a possible phylo-
genetic relationship. In the light of the intimate relationship
revealed by the more critical studies of the later investigators, all
green fluorescent bacteria are now classified in the Pseudomonas
group of Migula. Moreover it is now recognized that many of
the described species are identical, while the characters of others
grade into each other so imperceptibly that present bacteriological
methods fail to demonstrate specific constant differences upon
which differentiation may well he based.

The members of the group are widely distributed. They
are almost universally found in the presence of putrefying organic
material, and are abundant in soil, water, and air. They are
recognized as one of the most common types of water bacteria.
vSchmelck (26:546) noted that the predominating organisms in
glacial waters were of this sort, and Harrison (12) and Belli (2 \
observed B. fluorcscens liqucfaciens in hail. Jensen (13:613)
states that the fluorescent organisms occur in butter, while Thdni
(30:623) noted their presence in fifteen samples of lemonade
which he analyzed. Griffon (11) claims that B. fluorescens lique-
faciens and B. fluorescens putrida are capable of producing wet
rots of certain vegetables. The former lias been reported as the
causal organism in a carrot rot. while tobacco anthracnose is
attributed to B. aeruginosas, which according to Griffon, is
synonymous with B. fluorcscens putrida. Tomatoes grown under
glass have also been attacked by a stem canker said to be
due to B. fluorescens. Although the majority of fluorescent bac-
teria are harmless, certain species are reported as pathogenic to
animals. It is therefore evident that the bacteria of this group
are capable of existing' under a variety of conditions.

Fluorescent bacteria play a leading role in the deterioration
of maple sap. Fresh drawn sap of the early "runs" is water clear
and has a clean sweet flavor, but with the advent of warmer
weather it becomes more or less cloudy and a disagreeable flavor

Micro-organisms ok Maple Sap

develops. Such sap is popularly called sour, and several typ

arc recognized among which the most common is the so-called
green sap. In all the samples of greenish or greenish-brown sap
examined, the predominating organisms were of the fluoresced
type, and inoculation experiments have repeatedly demonstrated
their causal relationship to this type of spoiled sap.

The initial infection of the sap is doubtless brought in from
the hark of the tree, by wind, and by dripping rain and snow-
water. Falling snow and rain also probably bring in some in i
(ion from the air. Fluorescent bacteria were demonstrated on the
tree bark as well as in the snow and surface water and in un-
washed buckets and spouts from the previous season. Lai
numbers of the organisms remain in the spout and buckets from
season to season, unless these are thoroughly cleansed by boiling

Such maple sap organisms of the fluorescent type as have
been previously reported upon (4:492) were recognized as mem-
bers of the Pseudomonas group of Migula and more or less closely
related to Pseudomonas fluorescens. Both the liquefying and
non-liquefying types were observed.

On account of the prevalence of this type of bacteria in
maple sap and their importance in the maple sirup industry, a
critical study of the characters of the group was considered im
portant. The discussion falls naturally under two heads:

(1) A preliminary investigation with 42 strains of green
fluorescent sap bacteria.

(2) A more exhaustive comparative study of seven repre-
sentative strains selected in the preliminary work and of six
known species.

PrELimin \ry Studies TIVon 42 Strains of" Green Fi,i ori i int

Sap Bacteria

isolation

The 42 strains of green fluorescent sap bacteria were selected
from several hundred cultures isolated from abnormal maple sap
secured in different parts of Vermont during four successive

:,i>4

l'-ri.LETlN 167

sugar seasons. The numbers of the strains selected, the dates
of isolation and the sources are as follows :

Strain Date of isolation

CXII,

4/4/09

Randolph.

CXV\

((

tt

CXXVII,

4/5/09

tt

CXXVIII,

4/12/09

»

CXXIX,

a

tt

CXXX,

tl

a

CXXXIII,

4/12/09

i i

CXXX IV,

( .

tt

cxxxv,

it

a

CXXXVII,

tt

tt

cxxxvin.

a

tt

CXXXIX.

a

a

CXL,

4/12/09

tt

CXLI,

"

it

CXLII.

it

tt

CXLIII,

tt

a

CXLV,

a

a

CXLVI,

tt

tt

CXLVII,

tt

tt

CXLVIII,

4/13/09

tt

OXLIX,

"

tt

CL,

it

a

CLI,

a

tt

CLII.

tt

tt

CLIII,

it

a

CLIV,

tt

tt

CLV,

"

tt

CLVI,

..

i<

CLVIII,

tt

tt

CLIX,

tt

it

CLXXVII,

tt

tt

CLXXIX,

a

it

5

4/27/07 E.

Montpelier

XVI.

2/1/08

Burlington

XXXIII.

4/11/08

Bethel

XXXVI,

tt

a

XXXVIII.

a

"

L,

4/18/08

Fairfax

LI,

Cf

a

LIII,

it

it

LIV,

4/18/08

tt

LVI,

tt

(i

Source

Sour tree, Orchard 1
Second sour tree, Orchard I.
Third sour tree, Orchard I.

Green sour sap employed in
making sirup 24 (p. 358).

Green sap, Orchard II.

Milky sap, various orchards.

Greenish-yellow sour sap from
same tree as sirup 26 (page
358).

Sour sap.
Sour sap.

Sour sap.

Sour sap, Orchard I.

Sour sap, Orchard II.

1 Doubtfully fluorescent.

Micro-organisms of Maple Sap 525

METHODS OP WORK

Throughout the studies great care was exercised to secure
uniformity of methods so that the results with the several strains
might be comparable. In general the methods recommended in

"Standard Methods of Water Analysis*' (1905) and in Smith's
"Bacteria in Relation to Plant Diseases" (27) have been followed,
but in certain respects it has been deemed wise to introduce varia-
tions. The most important of these is the substitution of
"Liebig-'s Extract of Meat" for beef infusion. Media prepared
with meat extract has been used in this laboratory for several
years and has always given satisfaction. It is believed that media
prepared in this way is more uniform in composition than that
from infusion and it also possesses the very decided advantage
of being- free from muscle sugar. Actively fermenting strains of
B. coli develop in it without producing- visible gas. The objec-
tions usually advanced against meat extract that it frequently
contains resistant spores, that it may have been treated with pre-
servatives, or that it may contain injurious by-products of bacterial
development, have not seemed to be well founded. In our ex-
perience there is never more difficulty in sterilizing meat extract
than in sterilizing- beef infusion and we have never been able to
discover indications of preservatives or injurious decomposition
products, the most sensitive organisms developing as readily upon
one type of medium as upon the other. When cultures are to be
employed in studying gas evolution or acid formation, muscle
sugar introduces a complication. Jt is the general custom to
remove this by cultivating an active fermenter in it for a few-
hours. This is objectionable and is certain to introduce decom-
position products to such an extent as to render the media quite
unfit for the development of certain sensitive species. There is
great need of synthetic substitutes for the beef media composed of
synthesized chemicals, but until they are developed we believe the
employment of a standard brand of meat extract has points of
advantag-e over infusion.

526 Bulletin 167

The details necessary for duplication of our media are given
below for the convenience of any one who may have occasion
to review the results. Unless otherwise noted, media were pre-
pared with a distilled water of high purity and adjusted against
phenolphthalein to a reaction of +10 Fuller's scale. Sterilization
was effected by exposing in the autoclave at 6 pounds pressure
for 15 minutes, except with milk, potato, gelatin, and carbohy-
drate and glycerin media which were steamed in the Arnold for
15 minutes on each of three successive days.

Nutrient broth. — Ten grams of "Witte's Peptonum Siccum"
and 5 grams of "Liebig's Extract of Meat" were added to one
liter of distilled water and cooked in a double cooker until dis-
solved (about 30 minutes), restored to volume, titrated and ad-
justed in reaction to +10 Fuller's scale, cooked in double cooker
20 to 30 minutes, boiled 5 minutes over the open flame, titrated
and adjusted in reaction if necessary, filtered and sterilized.

Agar medium. — -This medium was prepared by adding 1.5',
of Bausch and Lomb agar flour to the nutrient broth, with sub-
sequent autoclaving, adjusting of reaction to +10 Fuller's scale,
and clearing with either fresh white of egg or dried albumen.

Gelatin medium. — Nutrient broth was stiffened with 12' i
of "Nelson's Photographic Gelatin No. 1" unless otherwise speci-
fied. The broth was heated, the gelatin added, steamed a few
minutes in the Arnold, adjusted to +10 Fuller's scale, cooled,
cleared with egg albumen, filtered, and sterilized by the inter-
mittent method.

REJUVENATION AND STOCK

The organisms were rejuvenated as recommended by trans-
ferring to nutrient broth, incubating 24 hours, transferring from
the young culture to a second tube of broth, incubating 24
hours again, and so on for three days. Plates were poured
from the third tube of broth to insure the purity of the
strain and transfers from the colonies were made to tubes of agar
and broth. The agar .and broth series was transferred monthlv

M [CRO ORGAN tSMS 01? M Al'l.K SAP

and from it, as a parent stuck, a sub stock was maintained. From
the sub-stock an inoculation stock was rejuvenated occasionally
and subjected to retransfer every 24 to 48 hours. Frequent
replating assured the purity of the strains. Unless otherwise

stated inoculations were made from 24 to 48 hour broth cultui
and incubation was at 25 ° C. Gelatin media were incubated at
200 C.

DETAILED FEATURES oi' Till'. FORTY-TWO STRAINS

The immediate object of the preliminary studies was to
separate the forty-two strains of fluorescent bacteria into groups,
in order that type organisms might be selected for a more ex-
haustive study. To this end attention was directed primarily
toward the characteristics which are expressed by the group
number, (Descriptive Card, Society of American Bacteriol-
ogists) ; but considerable additional data was secured on the
morphological, cultural, physical and biochemical characters of
the organisms. Therefore the discussion of the preliminary work
follows in a general way the detailed features as outlined in the
Society Card. For the group numbers of the strains and the
method of separating the series into groups, see pages 550-551.

I. Morphology

1. Form. — The organisms are all motile rods. I 'reparations
from 24 hour agar slant cultures of all -trains stained by Lowit's
method for flagella showed that some strains typically have one.
and others three or six, polar llagella, chains frequently exhibiting
numerous long flagella in a polar tuft. Strain CXV also has
lateral flagella (c. f., pp. 546, 555).

2. Grouping. — Short chains of two cells were typical but
longer chains were not uncommon. A surface scum or pellicle
was usually present on liquid media.

3. Motility. — Rapidly and quite persistently motile.

4. Bndospores. — None were demonstrated with stains or
by thermal death point determinations. Several common spore

528 Bulletin 167

stains were applied on material from many media and, except for
polar bodies, were without result.

5. Capsules. — Broth cultures of the three strains, XXXIII,
CXY, and CXLA , were commonly stringy. Capsules were demon-
strated on these strains, both by Welch's method and by Richard
Muir's contrast stain. Attempts to demonstrate capsulation on
some of the other strains were unsuccessful.

6. Stains. — All strains, except CXV, were Gram negative,
though decolorization was not always complete, granulations in
the rods and polar bodies being common. Strain CXV retained
Gram's stain after 4 minutes in absolute alcohol.

. Iqueous anilin stains. — Cells of all strains were easily stained,
but gave up the dye readily on washing-. The rods often showed
granulation or a bi-polar effect, certain portions seeming to have
a greater affinity for the stain than others.

II. Cultural Features

1. Agar stroke. — Growth abundant, filiform to echinulate,
spreading below; raised, smooth, glistening and somewhat viscid
or slimy. Medium more or less green fluorescent with ah strain-.
Strain CXV showed at most only a doubtful fluorescence.

2. Potato. — Growth moderate with all strains. A rather
narrow, smooth, filiform, light brown streak, becoming thick and
spreading later. Medium sometimes greened but not invariably.
Strain CXV often showed a dull rhizoid growth.

3. Agar stab. — Best development at top of puncture but
considerable sub-surface growth later. Filiform, becoming vil-
lous. Varying degrees of green fluorescence. Long crystals
were common in old cultures: they occurred under the surface
growth and about the puncture and were never detected in control
tubes. Similar crystals are illustrated by Smith (27:66). Beau-
tiful green fluorescence, varying somewhat in intensity with the
various strain-. Strain CXY was doubtfully fluorescent.

4. Gelatin. — Nutrient broth, stiffened with 10. 12, and 15' >
gelatin, was used. Several complete sets of the strains were

Micro-organisms of Maple Sap 529

tested out at 200 C. with similar results. The growth was best a<
the top with the puncture beaded to filiform. Restricted develop-
ment occurred along the puncture below the liquid gelatin.
Crateriform liquefaction becoming stratiform was typical of the
group. The reaction began in from i(> hours to 3 to 5 months.
Several weeks or longer was required to complete the liquefaction
of 7 cc. of the medium. Fluorescence was not marked, old cul-
tures showing it only faintly.

One set of cultures in duplicate held under observation over
eight months yielded noteworthy results. Seven strains began a
typical liquefaction after from three to five months, but the time
required for complete liquefaction of 7 cc. was not determined.
At the end of S>y2 months control plates were poured and the
organisms found to be present in pure culture. The organisms
recovered were fluorescent and capable of producing hydrogen
sulphid, the latter property being- peculiar to these seven strains

5. Nutrient broth. — A surface scum or a thin membranous
pellicle, often wrinkled, was usually present. Clouding was strong
at first, the medium becoming yellow-green fluorescent and clear-
ing with a deposition of a more or less coherent sediment.

6. Milk. — Fresh centrifuged milk reacting +12 to -+14
Fuller's scale, and sterilized by the fractional method, was used.
In from one to ten days a watery layer appeared at the surface
with a jelly-like thickening and greenish discoloration of the
substratum in all cultures except those of the following strains :
CXL, CXLL CXLII, CX1J11. CXLVI, CXLYII. XVI,
XXXYI, L, LI, and LIV. After 35 days- incubation, CXLVII,
XVI, XXXYI. XXXVIII, LI, and LIV showed doubtful clear-
ing. Strains CXL, CXLI, CXLII. CXI J II. CXLVI, and L in
all trials remained unchanged for about 3 months, when a very
slow digestion appeared.

7. Litmus milk. — Litmus milk was prepared by the addi-
tion of 2c/r of a saturated solution of chemically pure litmus to
fresh centrifuged milk and which was then sterilized by the inter-
mittent method.

530 Bulletin 167

Without enumerating the details of the minor differences
observed with the several strains the characteristics on this
medium may be summarized as follows: Alkali production and
more or less complete reduction of litmus occurred with all strains.
With the exception of strains CXL, CXLL CXLII, CXLHI,
CXLVI, XVI, XXXIII, XXXVIII, L, LI. and LIV, alkali was
produced promptly in the surface layer which soon cleared,
changed to acid and became more or less green in color. The
alkali layer diffused slowly downward, several well defined strata
often being differentiated. Digestion proceeded slowly with the
enumerated strains, and the medium gradually became more and
more blue, changing to purple at the time digestion became
evident.

8. Gelatin colonics. — A rather superficial examination of
the colonies of all strains on gelatin showed that they were very
much alike. Liquefaction occurred quickly in all strains except
CXL, CXLI, CXLII, CXL1II, CXLVI, CXLVIL XVI, XXX! 1 1.
L, LI, and LIV. For complete description of the types of colo-
nies, see detailed characteristics of individual strains, pages 567-

9. Agar colonies. — There were no essential differences
between the agar colonies of the several strains. For descrip-
tion of types, see detailed characteristics of individual strains
pages 568-570.

10. Cohn's nutrient solution. — Tins medium was made ac-
cording to the following formula:

Distilled water, 1000. cc,

Di-potassium phosphate, 5. gr.

Magnesium sulphate, 5. "

Ammonium tartrate, 10.

Potassium chlorid, 0.5 "

All strains were tried on this medium several times, but
only two strains, LI and LIV, showed growth. These grew
well with fluorescence, and the growth was accompanied by the
formation of crystals of magnesium ammonium phosphate. The
development was gradual but persistent, fluorescence and crystals

Micro-organisms of M\ru, Sap 531

appearing in from 15 to 23 days. Those strains showing no
growth were transferred to broth after twenty (lavs. The trans
fers developed normally becoming fluorescent in two days.

11. Uschinsky solution. — The medium was prepared as
follows :

Distilled water, 1000. cc.

Glycerin (Merck's Blue Label), 10. "

Sodium chlorid, 5. gr.

Calcium chlorid, 0.1 "

Magnesium sulphate, 0.4 "

Di-potassium phosphate, 2.2 "

Sodium asparaginate, 4. "

All strains except CXV developed well <>n this medium,
showing strong fluorescence, and a viscid pellicle and sediment.
Transfers made from tubes of CXV on Uschinsky solution de
veloped well in broth.

12. Nitrogen requirements. — For determining the nitrogen
requirements, a modified Uschinsky solution prepared as follows
was used as a basal medium.

Distilled water, 1000. cc.

Glycerin (Merck's Blue Label), 35. "

Sodium chlorid, G. gr.

Calcium chlorid, <».l "

Magnesium sulphate, 0.35 "

Di-potassium phosphate, 2.

The mixture was warmed to secure complete solution and
divided into three portions, one of which was reserved as a con-
trol while the others were treated respectively with 0.5', of c. p.
urea and 1% of asparagin.

No growth, or only a slight trace, occurred in the contn<l
medium. The feeble trace of transient growth in a portion of
the controls was attributed to the traces of nitrogen known to
be present in the glycerin.

Upon the urea medium all showed good growth, except
strain CXV which failed to develop. Transfers of this strain
from the urea medium to broth developed promptly.

582 Bulletin 167

Eight strains developed with fluorescence upon the asparagin
medium. These were as follows: CXXIX, CXXXIII, CXLVIII,
CXLIX, CLIX, CLXXYII. 5 and LVI. Transfers to nutrient
broth from the asparagin cultures of those strains which failed
to grow developed promptly.

III. Physical and Biochemical Features

1. The action of the organisms upon dextrose, lactose,
sucrose and glycerin. — The carbohydrate and glycerin media
were made by adding 2% of the sugars and 5% of glycerin
respectivelv to bouillon which was sugar free as shown by the
B. coli test. Cultures were observed for acid and gas produc-
tion and for growth in the closed arm of the Smith tube.

Cultures and controls were analysed in duplicate for acid
production after 1, 2, 4, 10, and 20 days incubation. Five cc.
of the material were pipetted into 45 cc. of distilled water in
Erlenmeyer flasks; j/2 cc. of 1% phenolphthalein solution was
added and the mixture boiled 2 minutes and titrated hot with
N/20 sodium hydroxid. Boiling often increased the amount
of free acid, probably through the volatilization of ammonia
formed simultaneously with the acid.

Control titrations were made upon bouillon cultures. Some
acid was detected which is thought to have resulted from the
decomposition of proteids, since muscle sugar was absent.
The conclusions as to acid production which are indicated in
the group number, pages 548 and 550, were drawn after com-
paring the results on the carbohydrate media with those on in-
oculated bouillon.

No gas was produced from the sugars or glycerin. Growth
in the closed arms of the fermentation tubes was limited and oc-
curred only in old cultures.

The detailed discussion of the characters on various car-
bohydrates follows :

Dextrose. — Acid production without gas evolution was char-
acteristic. Two '< dextrose litmus agar developed an acid

Micro-organisms of Maple Sap

reaction in from [8 hours to 4 days. Two '. dextrose bouillon
in fermentation tubes, titrated hot with N 20 sodium hydroxid
against phenolphthalein showed acid production, considerably
more being present than in sugar tree bouillon. 1 See tables
qg} c^o, 51). Previous to boiling, the acid was masked by tin-
ammonia produced, the cultures became pink on addition of
phenolphthalein but decolorized on boiling, with increased
acidity.

Lactose. — Litmus milk showed an alkaline reaction, becom-
ing doubtfully acid after several weeks. No acid, or a trace
in a few cases, was found by titrating 2(/i lactose bouillon.
(Tables 52, 53). By comparison most cultures in lactose bouil-
lon were found to produce no more acid than the control cul-
tures in sugar free bouillon.

Sucrose — Same as lactose (table 54)

Glycerin. — Acid production without gas formation was
observed with 11 strains on bouillon containing 50; of "Merck's
Blue Label" glycerin. (See tables 55, 56).

The figures in the tables represent the percent of normal
acid or alkali produced in the respective media and were cal-
culated by subtracting the average reaction of duplicate con-
trols from the average reaction of duplicate cultures, or vice
versa.

534

Bulletin 167

TABLE 4!). ACID AND ALKALI PRODUCED IN SUGAR FREE NUTRIENT BOUILLON

Test tubes, 10 cc.

Strain

3 (

lays

111 (

lays

number

Acid

Alkali

Acid

Alkali

ex 11,

0.30

0.0G

CXV,

0.09

CXXVII,

0.10

0.04

CXXVIII,

0.05

0.28

CXXIX,

11.10

0.42

(XXX,

0.02

0.26

CXXXIII,

ii.LM

OXXXIV,

0.2]

0.15

ex xxv,

0.3G

0.22

CXXXVII,

0.31

(1.12

cxxxvni,

0.29

0.1G

CXXX IX,

0.18

n.27

CXL,

11.:::,

0.36

CXL1,

0.29

0.49

CXLII,

li.l::

0.50

CXLIII,

0.25

11.02

CXLV,

11.11::

0.94

CXLVI,

0.30

n.no

CXLVII,

(1.17

0.79

CXLVIII,

0.20

0.41

CXLIX,

0.2]

0.48

CL,

0.20

CLI,

0.32

0.2a

CLII,

0.35

0.49

CLIII,

0.03

0.55

CLIV,

(1.14

0.16

CLV,

0.14

11.07

CLVI,

II. IS

0.09

CLVIII,

11. i::

0.34

CL1X,

0.28

0.21

CLXXVII.

it.:::!

0.03

CLXXIX.

0.14

11.22

5,

II. is

0.25

XVI,

0.4::

0.04

XXXIII,

0.14

0.51

XXXVI,

0.51

0.6S

XXXV11I,

0.0::

0.47

L,

0.36

0.36

I.I,

0.16

0.59

LIU,

(I. 4 I

0.72

LIV,

0.11

0.11

LVI,

II. Ill

0.74

,Mn ri i ORG ^\ ISMS 01? M \n r: S \r

lABLE 50 A( Mi AMI ALKALI PRODUCED IN 2'. UKXTKO I BUUIL1

Fermentation tubes

Strain

1

day

2 days

1 days

L0 days

20 d a j

number

Acid

A Ik.

Acid Alk.

Arid Alk.

Arid Alk.

Arid Alk.

(XII,

1.36

2.15

2.36

2.20

l.r.o

cxv,

0.00

n. is

0.59

0.48

0.70

CXXVII,

1.16

1.53

1.44

1.20

1.10

( 'XXVI 1 1,

0.53

1.08

0.92

0.84

0.71

ex xix.

L.00

l.ss

1.00

L.66

2.13

ex xx.

ii.Ts

n.lij

0.96

0.65

L.19

CXXXIII,

o.oo

0.63

0.76

0.60

0.80

(XXXIV,

0.00

0.56

0.60

0 Us

0.13

(XXXV,

0.38

0.32

0.38

1.72

0.63

CXXXVII,

0.24

0.28

0.79

2.10

Mil

(XXX VI II,

n. m;

II. (HI

0.10

1.19

0.08

( 'XXX IX,

0.48

0.5]

0.43

1.72

0.62

('XL,

1.21

1.10

1.42

2.63

i.::s

ex li.

1.06

I . I ::

1.02

2.34

I.OI

CXLII,

1.01

1.20

0.97

2.27

L.13

CXLIII,

1.31

1.39

1.55

2.67

L.35

CXLV,

0.66

1.11

1.30

2.55

1.11

CXLVI,

0.80

0.99

1.04

1 . 1 ::

1.01

CXLVII,

0.57

0.66

0.56

0.83

0.87

CXLVIII,

ii.t:;

1.2S

1.41

L.55

1.89

CXLIX,

n.Il

ii.:::,

1.21

2. us

2.31

CL,

II. SI

1.13

1.14

1.2:,

1.17

CLI,

0.42

0.45

0.44

ii.::::

0.7'.'

CLII,

0.46

0.27

0.28

0.29

0.27

CLIII,

0.22

0.46

u.!i 1

1.25

L.13

CLIV,

0.32

0.40

0.78

0.73

1.33

CLV,

0.30

0.52

(i.!»::

0.20

CLVI,

0.30

0.38

0.60

0.40

CLVIIT,

0.35

0.60

1.07

0.55

CLIX,

0.21

0.82

0.98

0.12

CLXXVII,

0.36

0.60

1.03

2.65

CLXXIX,

0.34

0.65

0.91

1.IO

5

U.7)

0.96

1.06

L.90

XVI,

0.70

0.78

0.68

L.58

XXXIII,

0.37

0.94

0.83

1.72

XXXVI,

1.28

L.64

L.51

2. 1 2

XXXVI 1 1,

o.or,

11.70

0.71

1.66

L,

0.31

0.40

0.20

1.10

LI,

0.04

0.50

0.49

L.28

LIII,

0.23

0.55

1.1 1

0.62

LIV,

0.00

0.58

0.51

1.68

LVI,

0.61

0.90

1.10

1.65

536

Bulletin 167

TABLE 51. ACID AND ALKALI PRODUCED IN -' > DEXTBOSE BOUILLON

Test tubes, 10 cc.

Strain

1

day

2 days

4 days

10 dayy

20 days

number

Acid Alk.

Acid Alk.

Acid Alk.

Acid Alk.

Acid Alk

CXII,

0.41

0.44

0.89

1.76

3.13

cxv,

0.03

0.21

0.28

0.71

0.75

CXXVII,

0.41

0.46

0.71

1.06

0.22

CXXVIII,

0.43

0.78

1.05

1.22

1.24

CXXIX,

0.48

0.55

1.54

2.23

2.50

cxxx,

0.41

0.80

1.15

1.44

1.4S

CXXXIII,

0.10

0.17

0.37

u.sr,

1.06

CXXXIV,

0.07

0.38

0.69

0.80

0.77

cxxxv,

0:17

0.18

o.L'::

0.36

0.99

CXXXVII.

0.10

0.16

ii.L'!"

LOS

1.16

CXXXVIII,

0.03

0.16

0.43

0.54

0.94

(XXXIX,

0.23

0.19

0.30

0.48

11. :,n

CXL,

0.87

1.06

1.66

1.52

1.61

CXLI,

0.58

0.81

1.13

1.35

1.71

CXLII,

0.34

0.91

1.24

1.30

1.51

CXLIII,

0.93

1.47

1.60

1.62

1.53

CXLV,

0.24

0.51

0.54

0.73

1.35

CXLVI,

0.39

0.64

1.02

1.40

1.55

CXLVII,

0.24

0.76

1.20

0.88

1.41

CXLVIII,

0.36

0.62

1.40

3.26

6.06

CXLIX,

0.26

0.98

1.50

2.32

2.44

CL,

0.16

o.Tl

1.03

2.17

2.33

CLI,

0.08

0.14

0.44

0.58

11.7s

CLII,

0.08

0.23

0.17

0.52

0.53

CLIII,

0.06

0.16

0.36

0.65

1.23

CLIV,

0.11

0.14

0.30

0.46

0.65

CLV,

0.00

0.19

0.29

0.46

1.91

CLVI.

0.00

0.18

0.2:.

0.55

0.83

CLVIII,

0.05

0.10

0.48

0.34

11. Ts

CLIX,

0.05

0.30

0.54

0.96

0.98

CLXXVII,

0.05

0.16

0.39

0.42

0.71

CLXXIX,

0.04

0.31

0.:,;,

0.92

1.32

5,

0.19

0.85

1.14

0.95

1.63

XVI,

0.22

0.59

1.01

0.73

1.15

XXXIII,

0.06

0.33

0.45

0.67

1.17

XXXVI.

0.18

o.S7

0.79

1.10

1.71

XXXVIII,

0.20

0.30

1.35

1.25

L,

0.04

0.14

0.36

0.42

0.93

LI.

0.04

0.32

o.ss

0.78

0.94

LIII,

0.17

0.11

0.29

0.86

1.43

LIV,

0.05

0.31

1.05

1.24

1.21

LVI.

0.23

0.41

0.55

1.17

1.61

Micro-organisms of Maple Sap ;':;.

TABLE 52. ACID AM) ALKALI PRODUCED in -' - LACTOSE BOUILLON

Fermentation tubes

Strain 1 day 2 days 4 days 10 days 20 'lays
number Acid Alk. Acid Alk. Acid Alk. Acid Alk. Acid Alk.

CXII 0.30 0.66 0.38 0.28 0.18

CXv' l,,,s 0.04 0.06 0.70 0.13

CXXVII 0.42 0.42 0.28 0.04 0.12

CXXVIII, 0.40 0.34 0.33 0.4:, 0.49

CXXIX 0.34 0.49 0.23 0.04 0.17

CXXX 0.22 0.24 0.18 0.01 0.37

CXXx'lII, 0.40 0.32 0.24 0.1C 0.05

CXXXIV. 0.40 0.40 0.60 0.62 0.31

cxxxv, °-23

CXXXVII, li:;1

CXXXVIII. 0.42

CXXXIX, " ::"
CXL,

CXLI, 1-57

CXLII, " ■•■-

CXLIII. 0.23

CXLV, 0.18

CXLVI, 0.32

CXLVII. 0.56

CXLVIII, 0.33

CXLIX, 0.54

CL, 0.46

CLI, 0.36

CLII, 0.42

CLIII. 0.36

CLIV. 0.34

CLV, 0.20

CLVI. 0.36

CLVIII, 0.50

CLIX, 0.45

CLXXVII, 0.29

CLXXIX, 0.14

5, 0.32

XVI, 0.11

XXXIII, 0.45

XXXVI, 0.25

XXXVIII, 0.19

L, 0.38

LI, 0.44

LIII, 0.34

LIV, n.fii'

LVI, 0.75

538

Bulletin 167

TABLE 53.

ACID AND

ALKALI PRODUCED

IN 2<2

3 LACTOSE BOUILLON

Test tubes, 12

cc.

1 day

2

days

4

days

10 days

20 days

Strain

Acid Alk.

Aci

d Alk.

Acid Alk.

Acid

Alk.

Acid Alk

CXII,

0.12

0.27

0.35

0.12

cxv,

0.12

0.11

0.15

0.28

CXXVII,

0.25

0.30

0.11

0.18

CXXVIII.

0.18

0.11

0.01

0.03

CXXIX,

0.21

0.03

0.17

0.35

cxxx,

0.17

0.07

0.19

0.32

CXXXIII,

0.20

0.07

0.21

CXXIV,

0.26

0.31

0.03

(XXXV.

0.29

0.2S

0.21

CXXXVII,

0.2G

0.32

0.35

(XXXVIII,

0.34

0.35

II. IIS

0.22

CXXXIX,

0.22

0.1!)

0.16

0.20

CXL,

0.12

0.12

0.3G

0.50

CXLI,

0.14

0.23

0.40

0.43

CXLII,

0.22

0.31

0.39

0.43

CXLIII,

0.13

0.19

0.28

0.54

CXLV,

0.18

0.20

O.il

0.31

CXLVI,

0.11

0.13

11.:::,

0.32

CXLVII,

0.24

0.26

0.34

0.53

CXLVIII,

0.2]

(Ml

0.11

0.29

M t< ri i i irg ^NISMS OiJ M \n.i-: S.\r

CABLE 54. ACID AND ALKALI PRODUCED IN 29? SUCROSE BO! Ml<>\

Test tubes, in CC.

1 day

o

days

4 i

lays

III 1

lays

20 i

[a j i

SI rain

A.i,| Alk.

Aci

(1 Alk.

Acid

Alk.

Acid

Alk.

Acid

All

(XII,

0.12

0.40

0.18

ii. 2 I

0.31

I'XV.

0.1 G

0.00

0.34

0.80

0.79

CXXV 1 1 ,

0.13

0.14

0.14

o.l 1

cxxvur,

0.04

0.21

0.03

0.06

CXXIX,

0.01

0.05

0.37

0.82

0.09

cxxx,

0.00

0.38

0.09

0.10

CXXXIII,

0.16

0.37

0.52

0.06

CXXXIV,

0.13

0.22

0.07

0.35

0.21

cxxxv,

0.22

0.19

0.19

0.33

0.19

(XXXVII,

0.19

O.IS

0.24

0.52

0.04

(XXXVIII,

0.28

0.30

o.l 1

ii L0

nil

( 'XXXIX,

0.26

0.22

o.2T

0.24

('XL,

0.10

0.18

0.32

0.56

0. ||

CXLI,

ii.ll

0.27

0.32

0.12

0.59

CXLII,

0.16

0.34

0.36

0.66

0.43

CXLIII,

0.16

0.18

0.29

0.59

0.59

CXLV.

0.23

0.2G

0.11

0.30

0.00

CXLVI,

0.04

0.21

0.48

0.52

n.72

CXLVII.

0.21

0.17

0.36

0.56

ii 67

CXLVI 1 1.

0.07

0.05

0.24

0.78

0.12

CXLIX,

0.11

0.05

0.12

0.14

o. I l

CL,

0.13

0.07

0.07

CLI,

0.07

0.16

0.16

0.12

O.IS

CLIL

0.17

0.16

0.17

0.03

0.08

CLIII,

0.09

0.09

0.15

0.24

0.18

CLIV,

0.08

0.01

0.03

0.44

CLV,

0.04

0.01

0.14

0.21

0.15

CLVI,

0.09

0.07

0.02

0.19

0.37

CLVIII,

0.13

0.14

0.31

0.07

0.06

CLIX,

n. hi

0.09

0.12

0.60

CLXXVII,

0.17

0.21

0.28

0.58

o.7o

CLXXIX,

0.07

0.08

0.23

0.58

0.19

5,

0.11

0.06

0.10

0.05

0.14

XVI,

0.07

0.26

0. 11

0.69

0.68

XXXIII,

0.10

0.16

0.26

0.31

0.37

XXXVI,

0.12

0.02

0.06

0.97

1.20

XXXVIII,

0.03

ii. 1 1

0.29

1. 2 I

1.17

L.

0.08

0.27

0.36

0.77

0.68

LI.

O.OO o.oo

0.20

0.40

0.71

0.63

LIII,

0.07

o.iiT

0.40

0.79

0.13

LIV,

0.02

0.21

0.31

0.72

n.72

LVI,

0.02

0.02

0.00

0.27

0.35

540

Bulletin 167

TABLE 55. ACID AND ALKALI PRODUCED I.N 5% GLYCERIN BOUILLON

Test tubes, 10 cc.

1 day 2

days

4 days

10 days

19 days

Strain

Acid Alk. Acid Alk.

Acid

Alk.

Acid

Alk.

Acid

Alk.

CXII,

0.00

0.25

1.35

1.56

CXV,

0.00

0.29

0.56

0.53

CXXVII,

0.06

0.00

0.62

0.94

CXXVIII,

0.13

0.04

0.47

0.26

CXXIX,

0.05

0.38

1.26

1.68

CXXX,

0.00

0.29

0.68

0.56

CXXXIII,

0.00

0.31

1.16

1.57

CXXXIV,

0.02

0.14

0.73

0.65

cxxxv,

0.15

0.07

0.39

1.10

CXXXVII,

0.12

0.18

0.90

1.36

CXXXVIII,

0.1 1

0.03

0.28

0.49

CXXXIX,

o.oo

0.20

1.16

1.06

CXL,

0.21

0.45

0.37

0.42

CXLI,

0.26

0.39

0.29

0.14

CXLII,

0.24

0.37

0.46

0.55

CXLIII,

It. 24

0.33

0.12

0.28

CXLV.

0.08

0.07

0.83

1.12

CXLVI.

0.27

0.44

0.52

0.23

CXLVII,

0.19

0.22

0.29

0.22

CXLVIII,

0.13

0.27

1.07

1.39

CXLIX,

0.00

0.26

1.39

2.08

CL,

0.03

0.54

1.43

1.05

CLI,

0.18

0.07

0.43

0.77

CLII,

0.13

0.04

0.19

0.33

CLIII,

0.02

0.34

0.88

1.31

CLIV,

0.00

0.16

0.70

0.96

CLV,

0.12

0.05

0.19

0.37

CLVI,

0.06

0.02

0.90

1.13

CLVIII,

0.12

0.11

0.49

0.67

CLIX,

0.18

0.26

0.98

1.69

CLXXVII,

0.19

0.20

0.19

0.34

CLXXIX,

0.14

0.60

1.00

1.35

5,

0.13

0.21

0.44

XVI,

0.27

0.17

0.00

XXXIII,

0.14

0.13

XXXVI,

0.15

II.IIS

0.26

XXXVIII,

0.20

0.51

0.52

L,

0.29

0.16

LI,

0.16

0.23

0.32

LIII.

0.28

0.95

1.06

LIV,

0.28

0.09

0.05

LVI,

0.27

1.38

1.81

Micro-organisms os Maple Sap

.11

TABLE 5G. ACID AND ALKALI PRODUCED IN •">' ; GLYCERIN BOUILLON

Duplicate trials Test tubes, 10 cc.

1 (

Jay 2 days 4

days

10

days 20 days

Strain Arid

Alk. Acid Alk. Aci

1 Alk.

Acid

Alk. Acid Alk

CXII,

0.30

1.08

CXV,

0.25

0.54

CXXVII,

0.04

o.o::

CXXVIII,

0.06

0.34

CXXIX,

0.34

0.86

CXXX,

0.35

0.51

CXXX1II,

0.25

0.67

CXXXIV,

0.32

0.86

cxxxv,

0.16

0.17

CXXXVII,

0.14

0.44

CXXXVIII,

0.05

0.02

CXXXIX,

0.05

0.32

CXL,

0.37

0.62

CXLI,

0.45

0.62

CXLII,

0.58

0.62

CXLIII,

0.37

0.62

CXLV.

0.08

0.14

CXLVI,

0.45

0.57

CXLVII,

0.49

0.43

CXLVI 1 1,

0.00

0.48

CXLIX,

0.05

0.86

CL,

0.25

0.58

CLI,

0.20

0.37

CLII,

0.20

0.11

CLIII,

0.0J

0.33

CLIV,

0.26

0.26

CLV,

0.1)8

0.02

CLVI,

0.15

0.44

CLVIII,

0.09

0.13

CLIX,

0.18

0.47

CLXXVII,

0.19

0.16

CLXXIX,

0.30

0.89

5,

0.30

0.20

XVI,

0.43

0.56

XXXIII,

0.18

0.20

XXXVI,

0.19

0.08

XXXVIII,

0.18

0.17

L,

(1.17

0.15

LI,

0.15

0.15

LIII,

0.14

0.59

LIV.

0.35

0.46

LVI,

0.60

1.23

542

Bulletin 107

2. Ammonia production. — Ammonia was produced by all

strains on nutrient broth, which is in accord with the statements
nf Thumm (31) (compare 20:754). The test was carried out
as follows: For the first of the series 125 cc. portions of nutri-
ent broth in Kjeldahl flasks were inoculated and incubated 10
days 111 the dark at room temperature (220 to 250 C). At the
end of this time determinations were made. Table 57 shows the
ammonia content of cultures and control in cc. of N/10 hydro-
chloric acid equivalent for the 125 cc. and it is also calculated to
a 100 cc. basis.

TABLE 57. AMMONIA PRODI I lloN

( Irganisin
number

(XII.
('XV.
CXXVII,
('XX VI II.
CXXIX,

exxx,

CXXXIII,

CXXXIV,

CXXXV,

CXXXVII,

(XXXVIII,

("XXX IX.

('XL,

CXLI.

CXLII.

Control,

1 KM equiva-

cc. N/10 HC1 equiva

:or 1 25 cc.

lent

for loo cc.

on 11

26.4

:■::.::

18.6

24.5

19.6

28.9

23.1

15.6

36.5

31.2

25.0

54.7

13.8

57.8

16.2

34.2

1^7.1

11.1

...» 1

34.8

27.8

34.6

7.6

6.1

9.8

7.8

L0.6

8.5

3.0

2.4

For the remainder of the series cultures were treated as
before except that i<><> cc. of media were used and the incuba-
tion period was 12 days at 200 C. The medium was identical
with that of the preceding series.

Micro-organisms of Maw.f. Sap

543

TABLE 58. AM \ln\iA PBODT ( TION

Organism

cc. X LO I in equiva-

Organism

cc.N/lo IK'l equiva

number

l.'in for LOO cc.

number

lenl for LOO cc.

CXLIII,

13.0

< '1. XX VII,

CXLV,

43.8

CLXXIX,

1*;.::

CXLVI.

9.5

5,

33.3

CXLVII,

13.8

XVI,

11.6

CXLVI 1 1,

39.4

XXXIII,

L7.2

CXLIX,

22.5

XXXVI,

14. 8

CL,

32.6

XXXVIII,

L0.7

CLI,

22.2

L,

7.8

CLII,

L4.2

LI,

7.7

CLIII,

48.1

LI 1 1,

49.0

CLIV,

44. G

LIV,

9.8

CLV,

2G.0

LVI,

G3.5

CLVI,

39.1

CXI I,

34.9

CLVIII,

44.7

cxv,

10.6

CLIX,

49.7

Control,

2.5

Control,

3.4

3. Action on nitrates. — Nitrates in nitrate broth were re-
duced to nitrites by three strains, as indicated by the iodo
starch reaction (27:63). All cultures showed ammonia with
Nessler's reagent ; but this was thought to have been an end
product of proteid decomposition rather than of nitrate reduc-
tion. No gas was produced.

Two formulas for nitrate broth were used with similar re-
sults.

I. Distilled water 1000. cc.

Liebig's extract of meat ::.

Witte's peptone 10. gr.

Potassium nitrate :!. gr.

II. Distilled water 1000. cc.

Witte's peptone 1. gr.

Potassium nitrate 2. gr.

Cultures in media of the first type were tested at the end
of 10 days and 30 days. Only strain LI responded with a posi-
tive reaction at the first test, but strains LI, LIV, and XXXVI
showed prompt blackening when tested after 30 days. The
reaction was more rapid in media prepared by formula If and
a positive reaction was obtained in 5 day old cultures of strains
LI, LIV and XXXVI. Only these three strains showed the
presence of nitrites at any time on either medium. The te I

544 Bulletin 167

were made repeatedly after incubation periods of various dura-
tion. The diphenylamine test for nitrates (32:340) gave a
positive reaction on cultures of all strains except XX X\ I.
(Compare 19:152-154).

4. Indol. — Indol was not detected in broth cultures of any
strain on the second or tenth day. The 10 cc. cultures were
tested with ten drops of chemically pure sulphuric acid and one
cc. of .02% sodium nitrite solution. Xo reaction appeared at the
end of ten minutes or after warming. (3:33).

5. Temperature relations. — The optimum temperature for
all strains was found to be in the neighborhood of 25 ° C. The
thermal death point was approximately determined in the pre-
liminary studies to lie between 490 and 550 C. The minimum
temperature for growth was below 30 C. except for strain CX\
which did not develop below 50 C. This strain showed growth
at 37° C. All others failed to develop at that temperature and
eighteen days exposure was fatal to them. Luxwolda (18)
states that B. fluorescens liquefaciens develops remarkably fast
at low temperature (30 to 5° C).

f>. Acids and alkalies. — A small amount of acid was pro-
duced in dextrose nutrient broth and indications of acid were
also observed in nutrient broth containing lactose, sucrose, and
glycerin. Titrations were made in hot solution with X/20
sodium hydroxid against phenolphthalein after 1. 2. 4. 10, and
20 days incubation. A trace of acid from proteid decomposi-
tion was observed with some of the strains in the sugar free
bouillon series.

Hydrogen sulphid. — Tubes of nutrient broth were pre-
pared in the usual way and, after inoculation, a strip of filter
paper moistened with lead acetate solution was suspended above
the culture in each tube. The paper in cultures of strain XVI
began to blacken at five days showing hydrogen sulphid pro-
duction. In ten days the papers in cultures of strains CXL,
CXLI, CXLII, CXLIII, CXLVI, XVI, LIV, CXV, showed
positive reactions, which in tin- cases of the last two strain.-,

Plate XIII. — Ps. fluorescens. Types of gelatin colonies: liquefaction

well under way. Figure 1. Strain LIII (three day old colony).

Figure 2. Strain XXXVI (four day old colony). (See pages 567-
568.) X 75.

Plate XIV. — Ps. fluorescens. Types of gelatin colonies. Figure 1.
Strain XXX (two day old colonies I. Figure 2. Strain L (two
old colonies). Figure 3. Strain LI II (two day old colonies i. (See
pages 567-568.) X 75.

Micro-organisms of Maple Sap 545

however, amounted to only a trace. After [9 days the papers
in CXV, CXL, CXLI, CXLII, CXUII, CXLVI, XVI, and

L showed considerable blackening while those in LI, Ll\ and
XXXVI showed but a trace. Test papers in the controls re-
mained unchanged.

Ammonia was produced by all strains, see pp. 542, 543.

7. Reduction processes. — Litmus in litmus milk and in
litmus asrars was somewhat reduced by all strains. Nitrates
were reduced to nitrites by three strains. (See action on
nitrates, page 543).

8. Odor. — A putrefactive odor was associated with de-
velopment on all the common media.

9. Gas production. — None observed by the Smith fermen-
tation tube method in dextrose, lactose, sucrose, glycerin, or
nitrate broths.

10. Crystals. — These were common in agar stab and plate
cultures, and in gelatin plate cultures. Twinned crystals of
magnesium ammonium phosphate were formed in Cohn's solu-
tion by strains LI and LIV.

11. Oxygen requirements. — Agar stab cultures were ex-
posed to an atmosphere freed of oxygen by Buchner's pyrogal-
lic acid method (33). The tubes were placed in liter Novy
jars containing 10 grams of dry pyrogallic acid, to which 150
cc. of 1% sodium hydroxid solution was then added and the
jars immediately sealed. These were opened at the end of two
weeks incubation at room temperature. A barely visible sur-
face colony and a slight growth along the puncture was ap-
parent with all strains. The growth was limited and without
fluorescence. While this property was apparent 24 hours later,
its failure to develop in the oxygen free atmosphere might be
attributed to the limited metabolism rather than to the absence
of oxygen. Repeated trials yielded confirmatory results, but
other methods (pages 588 and 589) have shown the organisms
to be strictly aerobic. The slight growth obtained by the pyro
gallic acid method is attributed to the presence of small amounts

546 Bulletin 167

of unabsorbed oxygen during the first hours of the experiment-.

12. Fluorescence. — The fluorescing property was possessed
by the strains in varying degrees. Some were never observed
to fluoresce with the intensity which was typical of others. The
degree of fluorescence was also variable with the same strain
upon different media. Strain CXV was never more than
doubtfully fluorescent, but it was included in the studies for
comparative purposes with the idea that its ability to fluoresce
might be intensified by proper cultural methods. This result has
not been obtained, however, and for this and other reasons it is
probable that the organism in question should not be included
with the fluorescent group.

The strongly fluorescent strains in general were the rapid
liquefiers and digesters, and they produced more ammonia than
the weakly fluorescent ones (c. f. chart page 551). Addition of
ammonia or sodium hydroxid generally brought out a bright yel-
low-green fluorescence in all vigorous cultures deficient in this
respect, with the exception of those of strain CXV (31). The
pigment disappeared in all cultures if made slightly acid, only
to reappear as soon as a slight excess of alkali was present.
In general upon agar and broth a beautiful yellow-green fluores-
cence appeared in from three to five days. On gelatin, fluores-
cence developed only to a slight degree.

The green fluorescent pigment. — The most extended in-
vestigations concerning the pigment formed by the fluorescent
bacteria have been made by Gessard (9), Thumm (.31). and
Jordan (14 and 15). Gessard concluded that B. pyocyaneas
produces two pigments, pyocyanin and fluorescine. In order
to bring out clearly the nature of the fluorescent pigment the
papers of Thumm and Jordan (14) are quite extensively cited.

Thumm studied the following species :
B. Iluorescens tenuis.
B. tluorescens putidus.
II. Iluorescens albus.
B. Iluorescens erythrospoms.

Micro-organisms of M \n.i. Sap 547

B. pyocyaneus < several strains i
Bact. syncyaneum.
B. viridans.

Mis results may be summarized as follows: All fluores
cent bacteria show in alkaline gelatin, first a sky blue, later a
moss green fluorescence and, accompanying' the latter, a yellow-
ing of the substratum. Old cultures with the exception of
those of B. fluorescens putridus are orange-red with dark green
fluorescence. All these colors are due to one yellow pigment.
When an acid producer and a fluorescent organism are culti-
vated together the yellow pigment is formed normally, but there
is no fluorescence. The green fluorescence in every case is
caused by the action of the ammonia produced. Calcium chlo-
rid is unessential for the formation of the pigment, but mag-
nesium sulphate and potassium phosphate are of the greatest
importance. Bact. syncyam um forms two pigments, a fluores-
cent one, and a steel-blue one.

Jordan (14) worked with the following specie-:

B. fluorescens albus.

B. fluorescens tenuis.

B. fluorescens mesentericus.

B. fluorescens putridus.

B. fluorescens liquefaciens.

B. viridans.
He summarizes his results as follows:
"Both sulphur and phosphorus are essential to pigment
formation but the nature of the base associated with these ele-
ments is not important. Other things being equal the presence
of the methyl or methylene group is coincident with superior
nutritive value and fluorescigenic power. Acid in the medium
not merely conceals the existence of the substance to which the
color is due but interferes with those vital activities of the bacilli
which in an alkaline medium lead to the production of that sub-
stance. An excess of substance favorable to growth and pig-
ment formation checks the latter though the former may be

548 Bulletin 167

greater than before." Jordan believes that the fluorescent prop-
erty is purely incidental. "When the medium contains cer-
tain compounds the metabolic activities of the organisms adjust
themselves to these conditions and it is of no physiological
significance that one of the products of this activity happens to
be fluorescent."

13. Diastasic action on potato starch. A thin starch
paste containing 2% thymol was added to equal volumes of
ten day old broth cultures, the tubes placed in the thermostat
for 8 hours, filtered and the filtrate tested with Fehling's solu-
tion. Sugar was detected in none of the cultures, indicating
that diastase had not been formed.

14. Group numbers. — The characteristics of the several
strains detailed above correspond as follows.

12 strains to the number, Pseudomonas 221.2332132

12 " " " " " 221.2332133

7 " " " " " 221.2322132

4 " " " " " 221.2322133

2 " " " " " 221.2333133

1 " " " " " 221.2323132

2 " " " " " 221.2222132

1 " " " ' " 221.2232133

1 " " " " Bacillus 221.2222?32

However since there is no sharp line between acid pn >-
duction and no acid production from the sugars and glycerin.
the only specific difference brought out by the group number is
in regard to nitrate reduction. If gelatin cultures had been hell
only six weeks to determine liquefaction, the group number oi
strains CXL. CXLI, CXLII. CXUII. CXLAT, XVI, and L,
would have been 222.2332133 and of strains LI and IJV 222.-
2333x33- For the group number corresponding to the respec-
tive strains, see page 550.

15. Separation of the 42 strains into groups. — The 42
strains naturally separate themselves into 6 groups with re-
spect to the following properties : the production of hydrogen
sulphid, the reduction of nitrates to nitrites, growth upon as-
paragin Uschinskv solution, Cohn's nutrient solution, gelatin.

Micro-organisms oi? Maple SAr 549

and milk. The separation of the strains into groups is shown
in the chart on the following page, where the relation existing
between ammonia production and fluorescence is also brought
out. In general the strains producing comparatively small
amounts of ammonia are the tardy liquefying and feebly fluores-
cent ones.

550

Bulletin 167
chart showing the separation

Group

Strain

number

CXXVII,

221.2332133

CXXVIII,

221.2332133

cxxx,

221.2332132

cxxxiv,

221.2332132

cxxxv,

221.2322133

CXXXVII,

221.2322132

CXXXVIII,

221.2332133

CXXXIX,

221.2322132

CXLV,

221.2222132

CL,

221.2332132

CLI,

221.2332132

CLIII,

221.2322132

CLIV,

221.2322132

CLV,

221.2332133

CLVI,

221.2322132

CLVIII,

221.2332133

CLXXIX,

221.2332132

XXXIII,

221.2332132

XXXVIII,

221.2322132

LIII,

221.2332132

CXII,

221.2332132

CXLVII,

221.2232133

CLI I,

221.2332132

CXXIX,

221.2322133

CXXXIII,

221.2332132

CXLVIII,

221.2222132

CXLIX,

221.2332132

CLIX,

221.2322132

CLXXVII,

221.2322133

5,

221.2322133

LVI,

221.2332132

XXXVI,

221.2323132

LI,

221.2333133

LIV.

221.2333133

CXL,

221.2332133

CXLT,

221.2332133

CXLII,

221.2332133

CXLIII,

221.2332133

CXLVI.

221.2332133

XVI,

221.2332133

L,

221.2332133

Hydrogen
sulphid

Nitrogen from
asparagin in
Uschinsky
solution

Nitrate

reduction

+
+
+
+
+
+
+
+

-I

+

+

cxv.

221.2222732

+
+
+
+
+
+
+

+

+

+, present; — , absent; ±, trace.

Micro-organisms of Maple Sap 551

of the 42 strains into groups

Ammonia in

100 cc. broth,

Growth

Gelatin

Digestion

10-12 days.

on Conn's

liquefaction

oi milk

.•<•. N/10 HCI

ii ut rienl

begins in

begins in

equivalent

solul ion

Fluorescence

24 hrs.

lOd.

19.6

—

strong

16hrs.

(id.

23.1

—

1 t

1G hrs.

6d.

25.0

—

. (

16 hrs.

10d.

46.2

. —

it

24 hrs.

Kid.

27.4

—

ii

24 hrs.

6d.

::::.i

—

it

G2 hrs.

LOd.

27.8

—

i*

24 hrs.

LOd.

34. •;

—

u

24 hrs.

10 (1.

43.8

—

• .

lGhrs.

6 (1.

32.6

—

If

40 hrs.

10 (1.

22.2

—

II

16 hrs.

10 d.

48.1

—

ii

16 hrs.

10(1.

44. G

—

u

20 hrs.

10 d.

26.0

—

ii

16 hrs.

10d.

39.1

—

u

1G hrs.

7(1.

44.7

—

ii

20 hrs.

t\<\.

46.3

—

tt

40 his.

lOd.

17.2

—

moderate

If, hrs.

Sd.

10.7

—

feeble

16 hrs.

Hid.

49.0

—

strong

10 d.

10 d.

26.4

—

"

30 d.

30 d.

L3.8

—

feeble

10 d.

10d.

14.2

—

moderate

16 hrs.

10 d.

36.5

—

st rong

16 his.

lOd.

43.8

—

tt

16 hrs.

10(1.

39.4

—

4*

LGhrs.

lOd.

22.5

— .

U

20 hrs.

8d.

49.7

—

•'

24 hrs.

Sd.

2::.:,

—

14

20 hrs.

Hid.

—

n

IG his.

5 d.

03.5

—

ii

24 hrs.

30 d.

L4.8

—

feeble

50(1.

30d.

7.7

+

>•

50 (1.

30 d.

9.8

+

a

5-6 mo.

3 mo.

7.0

—

Ceeble

5-6 mo.

:; mo.

9.8

—

tt

5 mo.

3 mo.

L0.6

—

a

4-5 mo.

3 mo.

L3.0

—

a

5-6 mo.

3 mo.

9.5

—

(i

4-5 mo.

40 d.

11.0

—

it

5 mo.

:: mo.

7.8

—

li

10 his.

10 d.

—

doubtful

552 Bulletin 167

Comparative Studies of Seven Representative Strains
Greex Fluorescent Sap Bacteria and
Six Known SpEcies.

After having- separated the 42 strains into groups in tin-
preliminary studies as shown above, 7 representative strains
were chosen for a more thorough study of their morphological,
cultural, physical and biochemical features. The strains selected
follow : From group one, strain CXLV, capsulated, and strain
CXII, a slow liquefier, non-capsulated ; from group two, strain
CXLVIII a rapid liquefier, non-capsulated and, in contrast to
the other groups, able to grow in asparagin Uschinsky solu-
tion; from group three, strain XXXVI, a nitrate reducer which
does not grow on Cohn's solution ; from group four, strain LI,
a nitrate reducer which grows on Cohn's solution ; from group
five, strain CXL-, a very tardy liquefier and casein digester,
producing abundant hydrogen sulphid, but not reducing nitrates;
from group six, strain CXV, a strong hydrogen sulphid
producer, a rapid liquefier, growing at 370 C, Gram positive,
and at most only doubtfully fluorescent. In addition, 6 strains
of known species were introduced for comparative purpose-
These are as follows :

Ps. alba t Zimmermann) Mignla.

Ps. iiuorescens (Flitgge)

Ps. longa (Zimmermann 1

Ps. mesenterica (Tataroff)

Ps. tenuis 1 Zimmermann 1

Ps. putrida (Fliigge)

The first five cultures were obtained from Krai's Labora-
torium, while the culture of Ps. putrida, added to the station
series some years ago, was supplied by Now.

The 13 strains were rejuvenated by preliminary cultiva-
tion, while frequent replating assured the purity of the cul-
tures. For methods and procedures see pages 525 and ?2i_). The
Societ} Card was used as a basis for the comparative studies
and, in general, as in the preliminary studies, the methods em-

M i» ri i organ isms i ii? .\' Ai'i.r. Sap

553

ployed were those recommended in "Standard Methods of Water

\u;il\ sis."

Detailed Features

I. Morphology

i. Vegetative cells. — Observations were made on 24 hour
agar hanging block cultures and on hanging drop preparations
from 16 to 24 hour agar slant condensation water. The cells
ot all strains were motile rods of medium length with rounded
ends, occurring typically in chains of two; less frequently singly,
or in chains of four or eight. Measurements were made of
what appeared to be individual cells, but in some cases the
figures given may represent double organisms. The limits of
size which were obtained are best shown in tabular form :

TABLE 59. LIMITS OF SIZE ON HANGING BLOCK

Maximum

Minimum

Strain

Length

Diameter

Length

Diameter

Ps. alba.

5.0]

nicrons

1.2 microns

1.7 microns

0.7 microns

Ps. fluorescens,

3.3

it

0.9

a

2.3

a

0.6

tt

CXII,

4.7

<(

1.0

a

2.0

it

0.6

tt

CXV,

2.6

it

1.0

tt

1.0

*'

0.8

it

CXL,

2.3

ti

0.9

a

1.0

ti

0.5

it

CXLV,

4.0

it

0.7

a

1.5

it

0.6

a

CXLVIII,

4.0

t t

0.6

tt

1.4

it

0.4

tt

XXXVI,

2.3

tt

0.7

it

1.6

tt

0.G

tt

LI,

3.3

a

0.7

tt

1.7

tt

0.5

tt

TABLE 60. LIMITS OF SIZE ON HANGING DROPS

Maximum

Minimum

Strain

Length

Diameter

Length

Diameter

Ps. alba.

2.5 microns

0.8 microns

1.25 microns

0.5 microns

Ps. fluorescens,

3.0

a

0.8

it

1.18

tt

0.6 "

Ps. longa,

2.2

tt

0.7

a

1.0

ct

0.6 "

Ps. mesenterica,

1.6

tt

0.8

a

1.05

t(

0.6 "

Ps. tenuis,

1.9

it

0.8

a

1.05

tt

0.5 "

Ps. putrida,

1.1

tt

0.7

a

0.9

tt

0.5 "

CXII,

1.5

tt

0.6

tt

0.9

ti

0.5 • "

CXV,

t.n

it

0.9

it

L.6

tt

0.5 "

CXL,

2.3

tt

0.9

tt

1.0

tt

0.5 "

CXLV.

3.0

tt

0.8

tt

0.9

tt

0.5 "

CXLVIII,

3.8

tt

0.8

ti

L.5

tt

0.5 "

XXXVI,

1.5

it

0.7

tt

0.7

tt

0.5 "

LI,

3.0

ti

O.S

a

1.2

tt

0.6 "

554 Bulletin 167

Measurements upon preparations from 24 hour agar slants
stained two minutes with carbol fuchsin without heating yielded
the following results.

TAKI.K

61. LIMITS OF

SIZE

(>N STAINED PREPARATIONS

Maximum

Minimum

Strain

Length

Diameter

Length

Diameter

P.s. alba,

2.8 microns

0.7 microns

1.1 microns

0.4 microns

Ps. fluoresceins,

2.1

tt

0.5

"

LI

a

0.4

t

Ps. longa,

1.9

a

0.7

"

1.3

• >

0.5

1

P.s. mesenterica

. 1.7

tt

0.7

it

0.9

tt

0.5

i

Ps. tenuis,

2.5

it

Q.8

a

1.7

tt

0.5

*

Ps. putrida,

1.1

ft

0.7

it

(1.9

"

0.5

1

CXI I,

2.2

u

0.G

a

1.:'

tt

0.5

1

CXV,

2.0

tt

0.8

> .

0.8

it

0.5

1

CXL.

1.4

a

1.0

tt

0.7

a

0.5

»

CXLV.

3.2

it

0.7

"

1.0

it

0.5

*

CXLVIII,

1M

• (

0.7

a

1.8

< .

0.6

*

XXXVI,

1.8

a

0.G

n

1.1

a

0.5

.

LI,

1.5

ti

0.G

u

1.1

i t

0.5 "

Fliigge (6:292) states that B. fluorcscens liquefaciens i^

.3-. 5 microns in diameter and 1-2 microns in length. According
to Mig'ula (21 :886) Ps. fluorcscens has the following dimen-
sions: diameter .68 microns; length 1.17-1.86 microns: Chester
(3:323) gives the following: 1-1.5 microns in length by .5
microns in diameter. Ellis (5:108) gives the length of the rods
from 1.5-6 microns and the diameter .4 microns. Zimmermann
(34:16) gives the following measurements for B. fluorcscens
tenuis. Length 1-1.85 microns; thickness approximately .8
microns.

2. Endospores. — Xo indication of endospores was ob-
served in any strain. The various spore stains which were
tried failed to show anything further than polar bodies or
granulation of rods. Zimmermann notes the presence of polar
granules in B. fluorcscens tenuis (34:16).

3. Flagclla. — Flagella preparations were made only upon
the sap bacteria. From 1 to 6 polar flagella were easily dem-
onstrated upon each strain except CXV. (See Plate X. figures
5 and 6). Preparations were made from 16 to 24 hour agar

Micro-organisms of Maple Sap 555

slants by means of Loerfler's method (with anilin gentian violet i
or by Lowit's modification of the same. The arrangement of
the flagella of the several strains was as follows.

Strain CXII. — A polar tuft of 3 flagella at one end of a
chain of two cells was typical. Occasionally tufts of 1 to 6
flagella occurred at both ends of double cells.

Strain CXI'.— This strain had peritrichiate flagella. Tufts
were common at each end of chains. Lateral flagella were com-
mon on single cells (c. f. Plate X, figure 2).

Strain CXL. — The flagella typically occurred in a polar tuft
at one end of the double cells, frequently at both ends.

Strain CXL]'. — Tufts of flagella were demonstrated on one
end of double cells, and occasionally on both ends. Single or-
ganisms usually had one polar flagellum.

Strain CXLVIII. — Double cells commonly had 1 or 3 flagella
at one end ; occasionally 1 at each end. Single cells with 2 polar
flagella were not uncommon.

Strain XXX]' I. — The double cells had 1, 3, or 6 polar
flagella, most commonly a tuft of 3 ; occasionally the flagella
occurred at both ends of the double cells.

Strain LI. — Most commonly 2 flagella occurred at one end
of the chains of two cells and 3 at the other, or 1 at one end
and 1 to 6 at the other. Double cells with I to 6 flagella at
one end were common.

4. Capsules. — Only the fluorescent sap bacteria were ex-
amined for capsules. Such an envelope was demonstrated upon
strains CXV and CXLV and upon strain XXXIII as previously
noted (page 528). A suggestion of capsulation was noted on
flagella preparations of strain CXLVIII, but the results with the
contrast capsule stain were negative. Cultures of strain XXXVI
(in 2% sucrose peptone solution were very stringy.

Strain CXV. — Capsules were easily demonstrated upon
preparations from 10 day broth cultures by either Welch's
method or by Richard Muir's contrast stain (22:106). Fre-

556 Bulletin 167

quently 2 and sometimes as many as 5 cells occurred side by
side in the same capsule, with flagella arising about the periph-
ery of the envelope (c. f. Plate X, figure 3).

Measurements obtained on the capsule preparations follow :

Length
of cell

l.G microns
1.8 "
1.8 "

Diameter
of cell

0.53 microns
0.53 "
0.50 "

Length of
capsule

2.18 microns
2.50 "
2.80 "

Diameter of
capsule

1.57 microns

1.57

1.82 "

Strain CXLV. — Material from 10 day broth cultures was
stained by Richard Muir's method and capsulation demon-
strated. Parallelism of cells within the capsules was absent
with this strain.

5. Zoogloca. — Not observed.

6. Involution forms. — Unusually long cells were common
in old cultures.

7. Staining reactions.

Gram's stain. — Films from 4 day agar colonies of the 13
strains were stained as follows: (22:103).

Treated with anilin gentian violet (22:101) i1/* minutes,
washed in water; treated with Gram's solution iJ/2 minutes,
and immersed in absolute alcohol 4 minutes. All of the fluores
cent organisms were Gram negative (21 :888). All strains
except Ps. alba showed some granular spots which failed to
decolorize in 4 minutes. Strain CXV remained deeply stained
even after 15 minutes in absolute alcohol.

Gram's stain with amyl alcohol (29:108). — Films of strains
CXII, CXV, and CXL were stained as above, but amyl alcohol
was used 5 minutes for decolorizing. Strain CXII retained
the stain faintly, some cells more than others, and a bi-polar
effect was noticeable. With CXV the stain was irregularly
retained, the cells having a granular appearance. Cells of
strain CXL were only partially decolorized even after 15
minutes exposure to amyl alcohol.

Micro-organisms of M \ n.i-: Sap 551

Aqueous fuchsia. — Aqueous fuchsin was prepared by dis-
solving one gram of Griibler's fuchsin in jo cc. of water,
films from 4 day agar colonies of the 13 strains were treated
with this stain for two minutes and washed in water. The films
were mounted in water and microscopic examinations made.
The cells of Ps. fluorescens, Ps. longa, Ps. tenuis, CXV, CXL,
and LI were deeply stained: while those of Ps. alba, Ps. mesen-
teric^ Ps. putrida, CXII, CXLV, CXLV11I. and XXXV] were
only faintly stained. The cells from all strains except CXV pre-
sented a granular plasmolysed appearance and with strains Ps.
tenuis and CXL a bi-polar staining was common.

Aqueous gentian violet. — The stain was prepared by dis-
solving 1 gram of Griibler's gentian violet in 10 cc. of water.
Preparations from 4 day agar colonies were stained 2 minutes
and washed in water. The cells of Ps. alba, Ps. longa, CXV,
CXL, CXLV, CXLVIII, and LI were well stained, while those
of Ps. fluorescens, Ps. tenuis, Ps. mesenterica, Ps. putrida,
XXXVI, and CXII were rather faintly stained. Plasmolysis
occurred with all strains but was particularly noticeable in the
following: Ps. fluorescens, Ps. longa, Ps. tenuis, Ps. putrida,
and CXII.

Carbol fuchsin {Ziehl). — Films were prepared from 4 day
agar colonies of all strains, stained two minutes with Ziehl's
carbol fuchsin and rinsed with water. Strain CXV was the
only organism in which the entire cell was stained. Cells of
Ps. alba, Ps. fluorescens, Ps. longa, Ps. putrida were only faint-
ly stained, while those of Ps. mesenterica, Ps. tenuis, CXII,
CXL, CXLV, CXLVIII, XXXVI, and LI were fairly well
stained but showed a bi-polar effect. Chains of cells of the lat-
ter two mentioned strains presented a barred appearance.

II. Cultural Fkatukks

1. Agar stroke. — On agar stroke the thirteen strains dis-
played similar growth characters, indeed 110 specific constant dif-
ference can he pointed out. The line of inoculation first ap-

558 Bulletin 167

peared in 24 hours, as a faint white streak, rapidly becoming
beaded above, moist, glistening and of slimy consistency. It
had a white to light grayish color and often appeared nacreous
in certain lights. The medium became more or less green
fluorescent on the third day, the coloration gradually increas-
ing in intensity. Strain CXV grew more slowly than the others
and with doubtful fluorescence.

2. Potato slants. — Cylinders were cut from large tubers
and divided so as to give a slant surface. These were washed
for several hours in flowing tap water before they were placed
in the tubes containing a few drops of distilled water. Sterili-
zation was accomplished by the intermittent method.

Upon this medium a moderate growth occurred with all
strains. At first in about twenty-four hours a very delicate
whitish accumulation could just be distinguished. This de-
veloped slowly into a light brown, slimy, smooth, filiform,
spreading band, later becoming thick and darker brown, some-
times even chocolate brown in old cultures. Strain CXV was
characterized frequently by a more or less corrugated to rhizoid,
slimy growth, moist at first, later appearing rather dry. The
medium was more or less grayed by all strains. Strong green-
ing of the substratum was present with some strains but not
constantly. This reaction was observed with the following
strains; CXII, in 31 days: CXLV, CXLVIII, LI in 5 days;
Ps. fluorescais in 3 days.

3. Loeftlcr's blood scrum. — This medium was not em-
ployed.

4. Agar stab. — Specific differential characters were not
found on this medium. The surface growth was moderate
with all 13 strains, at first white, becoming more or less brown,
round and entire to contoured with toothed edge. The punc-
ture growth which was at first beaded to filiform became echin-
ate to villous, the latter character being typical of old cultures.
The best development occurred in the upper portion of the stab.
The medium was at first blue-green fluorescent, becoming more

Micro-organisms of Maple Sap 559

and more yellow-green. In the old cultures the agar had as-
sumed a brownish to amber color. Fluorescence was not as
strongly evidenced by some strains as by others. The strongly
fluorescent strains were: Ps. fluorescens, CXLVIII, CXII,
CXLV, Ps. mesenterica, CXL, Ps. tenuis and LI named ap-
proximately in the order of their fluorescing powers. Ps. alba,
Ps. longa, Ps. putrida, and XXXVI showed moderate fluores-
cence. Strain CXY was at most doubtfully fluorescent.

5. Gelatin stab at 20° C. — Gelatin media were prepared
with "Nelson's Photographic Gelatin No. 1," and a "Gold Label"
gelatin. Sometimes 10 and sometimes \ 2' < were employed.
Neither the percent of gelatin nor the brand was observed to
have any significant effect on the cultural characters. ( )n gela-
tin media there were two types among the 13 strains, a liquefying
and a non-liquefying type, the latter including two very tardy
Iiquefiers. However, a sharp division line between types does
not exist.

Liquefying type. — To this group belong Ps. fluorescens, Ps.
mesenterica, CXII, CXV, CXLV, CXLVIII, and XXXVI.

Ps. fluorescens. — In 18 hours a slight growth appeared as a
filiform whitish line. Crateriform liquefaction began in two days
gradually extending the shallow crater until the liquefaction
assumed a stratiform type. The fluid was at first yellowish white
and moderately turbid, the stab being filiform and finely villous
above, beaded and finely villous below. A slight green fluo-
rescence was apparent, but only in the liquid portions. The
fluorescence in gelatin, -\-io Fuller's scale, was never as marked
as in agar of the same reaction. The influence of the reaction
upon fluorescence is apparent from the results of two series of
gelatin cultures, one of which had an initial reaction of — 4.7 Ful-
ler's scale and the other of — 12. The fluorescence appeared
earlier and became noticeably more intense in the more alkaline
medium, showing that a slightly alkaline gelatin is more favora-
ble to the development of fluorescence than a neutral or acid one.
This is in accord with the statements of Thumm (31) and Tor-

560 Bulletin 167

dan (14). During- the incubation the alkali was partly neu-
tralized by the carbon dioxid absorbed from the air. The actual
reaction at the time the notes were taken was determined by
titrating control tubes with N/20 sodium hydroxid and phenol-
phthalein. The medium originally reacting at — 4.7 Fuller's
scale had an actual reaction of +2.4; that originally reacting at
— 12.4 had an actual reaction of — 6.1. Fluorescence had not
appeared in 26 days in the medium having an original reaction
of — 4.7, but in the medium having an original reaction of — 12.4
fluorescence was as marked as in agar cultures. The actual reac-
tion of the more alkaline medium at this time was -j-2.7 Fuller's
scale. Liquefaction has proceeded farther in this medium than
in the other, which now had a reaction of +4.4 Fuller's scale.
Gelatin liquefaction proceeded slowly and was completed in
45 days, at which time the fluid was rather clear and of an olive
green color. Considerable white flaky precipitate was thrown
down as liquefaction proceeded and a scum usually appeared.
There was a slight development in the line of puncture, liquefac-
tion occasionally appearing as a tunnel or as beaded spots along
the stab.

Ps. mesenterial. — In 1 or 2 days the growth appeared as a
white line, filiform above and beaded below. The colony was
white with depressed center and irregularly dentate edge, occa-
sionally round with entire edge. Crateriform liquefaction usually
began in from 8 to 17 days, but was occasionally delayed until
about a month had elapsed. The liquefaction gradually became
stratiform and was practically completed in about 4 months. A
somewhat more rapid liquefaction and stronger fluorescence oc-
curred in alkaline gelatin ; however traces of green fluorescence
could be detected occasionally in gelatin reacting +10 Fuller's
scale.

Strain CXIL — At the outset of these studies this organism
hist began gelatin liquefaction in about 10 days : at their close,
after an interval of two years, gelatin liquefaction constantly
appeared in 24 hours. In one to two days the growth was a

Micro-organisms Or' Maple Sap -''''I

while filiform line which gradually became echinate to villous.
Liquefaction proceeded from crateriform to stratiform and was
completed in about one month. Fluorescence was rarely seen in
-j-io gelatin but appeared in alkaline gelatin (compare with Ps.
mesenterial and Ps. fluoresceins pages ^'?n, 560). An abundant
white flak}- precipitate settled out as liquefaction proceeded.
Growth in the puncture was transient, the line of inoculation re-
maining about the same.

Strain CXV. — Growth appeared in a few hours, the puncture
being beaded, liquefaction becoming evident in [6 hours. The
colony sank in a saucer-like depression, the liquefaction soon
becoming stratiform, proceeding quite rapidly at first but grad-
ually slowing down when nearly all the 7 cc. of medium had been
liquefied. The line of puncture quite frequently showed con-
siderable liquefaction. The fluid was slightly turbid but devel-
oped neither yellow nor green color. A thin surface membrane
developed and a moderate amount of white precipitate was thrown
down. Liquefaction of the 7 cc. of medium was completed in
about a month's time.

Strain CXLV. — This organism at the beginning of the studies
was one of the most rapid liquefiers, commencing liquefaction in
24 hours and completing it in 27 days : but in the course of 18
months' cultivation it has gradually lost its ability to liquefy
gelatin reacting -f-10 Fuller's scale. Otherwise it does not differ
essentially from strain CXII. In the studies with alkaline gela-
tin media with original reactions of — 4.7 and — 12.4 Fuller's scale,
this organism showed crateriform liquefaction in 26 days in the
— 12.4 medium and none in the — 4.7 medium. At this time the
— 12.4 medium had a reaction of -f-2.7 Fuller's* scale. It is thus
evident that this organism retained the power to liquefy alkaline
or very slightly acid gelatin, after it had practically lost it-
ability to liquefy the ordinary gelatin. Fluorescence occurred
only in the alkaline gelatin.

Strain CXLVIII. — This organism resembled l's. fluorescens
very closely on gelatin stab, but in old cultures it did not show

562 Bulletin 167

the olive green color noted with the latter organism. No other
points of difference were observed.

Si rain XXXVI. — This strain could not be distinguished from
Ps. fluorescent or strain CXLVIII, except by a more feeble fluo-
rescence on alkaline gelatin.

Strain LI. — This organism also resembled Ps. fluorescens
closely. It could be distinguished only by the fact that liquefac-
tion was delayed, first appearing in about 50 days. LI is evi-
dently a strain of Ps. fluoresceins, tending toward the variety iion-
liqucfaciens.

The non-liquefying type. — To this group belong the follow-
ing organisms: Ps. putrida, Ps. alba, Ps. longa. Strains Ps.
tenuis and CXL are classified here provisionally since liquefaction
is long delayed. Cultures were held under observation for six
months in a moist chamber at 180 to 200 C.

Ps. putrida. — Growth was slow, becoming visible the fourth
day ; the stab was filiform above and beaded below with a small
white colony at the surface. At 12 days the colony was round
and brownish, the surface being' somewhat contoured. At 20
days the colony had become chocolate brown at the center, and
the edge showed spiny outgrowths. In from 20 to 40 days the
medium showed a strong browning, beginning at the surface and
progressing slowly downward. No liquefaction was observed in
five months. At this time the colony was round, about 1 cm. in
diameter, having a reddish-brown center and raised edge with
rhizoid and spiny processes; the puncture was beaded and villous.

Ps. alba and Ps. longa. — Cultures were similar to those of
Ps. putrida, but no browning of the colony or of the medium
was apparent. In 5 months the following characters were in
evidence; colony, white, round with beaded to lacerate edge:
puncture, beaded and widely villous in the upper portions; no
liquefaction.

Ps. tenuis. — Young cultures were not essentially different
from those of Ps. alba and Ps. putrida. Some colonies were
round and entire, while others were erose. The stab was filiform

Micro-organisms of M ai'i.k Sap 563

to beaded, becoming villous later. Liquefaction constantly began

after about 4 mouths and proceeded slowly, the fluid being thick
and slimy. At this time the stab was a faint white filiform line,
development in this portion of the culture having ceased. Ac
cording to Migula's classification Ps. tenuis is a non-liquefier.

( 21 \(J\o).

Strain CXL. — Cultures of this organism were like those of
Ps. tenuis. Liquefaction constantly began after about 5 months'
incubation. At this time the stab was either filiform and beaded.
or filiform, beaded, and widely villous. As in the cultures of Ps.
tenuis the liquid was quite thick and slimy. The purity of the
culture and its identity with the original strain was proved. It
is worthy of note that this organism, when held at 25 s C. pep-
tonized milk only after three months' incubation.

6. Nutrient broth. — Upon this medium slight difference-
were observed among the different strains. Moderate clouding
appeared in 18 to 24 hours in all except CXV, which was rather
tardy in showing growth in most liquid media. The medium
rapidly became more or less turbid, forming a scum or pellicle
which at intervals settled to the bottom of the tube as a white
flaky to viscid precipitate. A rather tough membranous pellicle
was produced by stain CXLY. More or less blue-green fluo-
rescence was apparent in from 3 to 10 days, but was never strong
in Ps. longa, Ps. tenuis. Ps. putrida or XXXVI, except in alkaline
broth. Strain CXV produced only a trace of fluorescence. The
other strains usually showed beautiful green fluorescence, but
it was not constantly present. The blue-green rapidly became
yellow-green and in old cultures the medium cleared and showed
an amber yellow color. Cultures of CXV and CXI A often
became quite stringy.

Strain CXI'. — A slight to moderate clouding appeared in
from 2 to 5 days, gradually increasing in intensity. Later the
cultures developed a white surface membrane, the substratum
becoming quite clear. An abundant white precipitate was thrown
down. Old cultures became amber colored.

5fi4

Bulletin 167

7. Milk. — The medium consisted of fresh eentrifuged milk,
titrated, filtered, and tubed, 10 ec. per tube. Sterilization was
effected by the intermittent method.

Acid production. — Triplicate titrations of cultures and con-
trols were made after 1, 2, 4, 10, 20, and 46 days' incubation.
Five cc. of cultures and controls respectively were pipetted into
45 cc. of distilled water in Erlenmeyer flasks, 0.5 cc. of 1-100
phenolphthalein added, the mixture boiled 2 minutes and titrated
hot with N/20 sodium hydroxid. The averages of the three
titrations of cultures were compared with the averages of those
of the three control tubes, in order to obtain the percent of normal
acid or alkali produced at each time. The results obtained were
as follows :

TABLE

62. ACID PRODUCTION IN

MILK

Strain

1

day

2 1

lays

4

days

10 days

20

days

Acid

Alk.

Acic

I Alk.

Acid All

Acid Alk.

Acic

[ Alk.

Ps. alba,

0.02

0.15

0.54

0.89

0.93

Ps. ftuorescens,

0.07

0.13

l

3.46

1.76

3.77

Ps. Jonga,

0.22

0.50

0.62

1.10

1.14

Ps. mesent'a.

0.00

0.00

0.30

0.69

0.87

1.11

Ps. tenuis.

0.26

0.31

0.46

0.75

0.83

Ps. putrida,

0.12

0.05

0.03

0.29

0.04

CXII,

0.20

0.81

1.28

2.80

3.45

CXV,

0.03

0.05

1

3.13

0.54

1.20

CXL,

0.04

0.08

0.10

0.38

0.72

CXLV.

0.29

0.28

0.48

0.44

0.90

CXLVIII,

u.m;

0.78

L.4G

2.42

3.70

XXXVI,

0.20

0.28

0.44

0.17

0.74

LI,

0.03

0.21

0.22

0.56

0.91

Strain

20

Acid.

davs
Alk.

46
Acid

davs
Alk.

Ps. alba

1.15

1.20

Ps. fluorescens.

5.29

4.41

Ps. longa,

1.03

1.22

Ps. mesenterica,

1.19

1.22

Ps. tenuis.

0.99

0.72

Ps. put riila.

0.0c

0.29

CXII,

3.54

4. I'll

CXV,

1.82

3.85

CXL,

0.62

0 -7

CXLV,

0.45

0.12

CXLVIII.

5.31

3.46

XXXVI,

0.72

2.20

LI,

0.57

1.22

Micro-organisms of M \ru Sap 565

There were _> types among the i,^ strains according to their
action on milk. One type produced considerable acid and i
agulated and digested the medium quite rapidly, a more or less
greenish color being present. The coaguhun was of a more or
less jelly-like consistency. The other type showed clearing with-
out coagulation of the milk, the action being delayed and taking
place very slowly. With certain strains digestion was not evi-
dent until about the third month. To the first group belong the
following strains: Ps. fluorescens, CXII, CXV, CXLVIII, and
XXXVI; and to the second group belong: Ps. alba, Ps. longa,
Ps. mesenterica, Ps. tenuis. Ps. putrida, CXL, CXLV, and LI.

Strains Ps. fluorescein, CXII, and CXLVIII of the first
type, behaved similarly on milk. A more or less jelly-like co-
agulation appeared in from 2 to 10 days coincident with acid
production. The coagulum formed was rather slimy with the
first organism named but with the others was quite firm. Diges-
tion with more or less greenish discoloration of the peptonized
portion was apparent in 4 days, becoming complete in from 18 to
30 days, the cultures at this time being relatively clear and closely
resembling broth cultures of similar age. A deep green color
appeared at the surface in these old cultures and upon agitation
the whole medium became olive green. The coloration was more
pronounced in Ps. fluorescens than in the other two. Luxwolda
(18) states that B. fluorescens liquefaciens first coagulates milk
with a lab ferment and then peptonizes it.

Strain XXXVI differed from the three preceding organisms
in that only a trace of green color was present. Coagulation with
this strain was always of jelly-like consistency.

Strain CXV showed clearing without coagulation until about
the eighteenth day when a firm coagulum appeared coincident
with acid production. Digestion continued and was completed in
from 30 to 104 days. The medium never appeared green but was
more or less straw colored throughout.

With the eight strains of the second type the medium re-
mained practically unchanged in appearance from ro to 85 days,

566 Bulletin 167

the only change which could be noted being- its scarcely percep-
tible browning or greening. Clearing without coagulation oc-
curred sooner or later with all strains, and was complete in from
1 in 5 months' time. Cultures of strain CXL, in particular could
not be distinguished from the control tubes until about 3 months
after inoculation, when clearing without coagulation began to be
apparent. This reaction was completed in from 4 to 5 months.
Repeated trials with recovery of the organism showed that this
clearing of the medium was not due to contamination. (Compare
action of this organism on milk with its action on gelatin j. The
only evidence of growth with strains Ps. longa, Ps. mesenterica,
and Ps. putrida until after a month had elapsed, was a scarcely
perceptible browning of the medium and a thin scum.

8. Litmus milk. — To freshly centrifuged milk with a reac-
tion of -(-to to -(-12 Fuller's scale was added 2% of a saturated
aqueous solution of Merck's chemically pure blue litmus ; the
medium was then filtered, tubed and sterilized by the intermittent
method. When ready for use the milk had a rich lavender color.

On this medium all strains developed an alkaline reaction.
which, when digestion commenced, was succeeded by one of an
acid character. The groups already referred to on gelatin and
milk were likewise differentiated here.

The first group, rapid liquefiers and rapid digesters, showed
an alkaline reaction at the outset which soon gave place to an
acid reaction when digestion commenced. As digestion pro-
ceeded, a purple to a reddish coloration advanced into the sub-
stratum, the latter becoming first blue, then purple and red,
finally bleaching to a straw or amber color when digestion was
complete. The surface layers were more or less greenish, espe-
cially with Ps. fluorescens, CXII, CXLVIII. Upon agitation of
the tubes when digestion was completed, the medium became
olive green throughout. To this first group belong Ps. fluores-
cens, CXII, CXV, CXLVITT, and XXXVI. Ps. mesenterica on
this medium reacts more like the second group.

Micro-organisms of Maple Sap 567

The second group, non-liquefiers, (and very tardy liqui
and digesters), comprises the following strains: /'.v. alba, Ps.
longa, Ps. putrida, Ps. tenuis. CXL, U and Ps. mesenterial.
Alkalinity was apparent the second day. except with Ps. putrida;
it first manifested this reaction on the tenth day. Alkalinity grad
ually increased at first in cultures of this group, and after some
time acidity developed simultaneously with digestion. After
several months, when digestion was nearly complete, cultures ol
all strains except CXL and Ps. putrida were colored purple
throughout, without perceptible clearing. Cultures of CXLwi
pink throughout, but otherwise resembled Ps. putrida.

The action of all [3 strains on litmus milk was essentially
the same; first, alkali production, then acid production accom-
panied by digestion.

9. Gelatin colonics. — The colony of strain CXL VII] is de-
scribed as the type of the rapidly liquefying group.

Macroscopically ; at first punctiform to round, yellowish,
raised, smooth, glistening, and entire, soon liquefying and sink-
ing in saucer-like depressions, the fluid showing slight green
fluorescence. Microscopically; round, entire, convex, brownish,
center dark and surrounded by a thinner zone (liquid). In-
ternally granular to grumose with spiny processes about the edge.
The filaments were variously oriented, being more loosely inter-
twined at the vd^v where they extended out into the liquid por-
tion. The colonies soon disintegrated, becoming floccose or
grumose masses in the liquefied gelatin. (See I'latc XII-XR I.

Ps. fluorescens and strains CXI I and CXI A' could not he
distinguished specifically from CXIA 111.

Strain CXV. — This strain closely resembles the type. Radiate
filaments were frequently present, often being almost perfectly
symmetrical, the ends of the filaments forming the bordering
fringe of the colonies. Liquefaction was very rapid.

Strain XXXVI. — At first round, entire, yellowish brown,
finely granular discs, sometimes with an irregular cracked appear-
ance. Some were undulately zoned and the surface irregularly

•>,;> Bulletin 167

marked with curved lines. The internal structure became
grumose, to broken and fimbriate. A slow and somewhat delayed
liquefaction began as a finely granular outer zone, the appear-
ance thereafter conforming to the type, CXLVIII.

Ps. alba is described as the type of the non-liquefying and
tardy liquefying group. Microscopically ; punctiform, raised,
smooth, yellowish. Microscopically: roundish, entire to broken.
yellow-brown, somewhat gray towards the edge. Internally
grumose to fluccose. Ps. putrida; slower growth than Ps. alba:
macroscopically like the latter. Microscopically; round, entire,
brownish, granular. The surface was slightly ridged and fur-
rowed giving a shadowy appearance to the colony. Later the
colonies became grumose to broken and were dark brown in color
Ps. tenuis. — Macroscopically; like Ps. alba. Microscopic-
ally; roundish, granular to grumose or floccose, becoming spiny
or fimbriate.

Strain CXL. — Macroscopically; like Ps. alba. Microscopic-
ally; much like the type, being round, convex, entire, yellowish-
brown, and granular toward the center, the edge being thicker
and somewhat grayish. Others were yellow-brown throughout:
larger ones were often roundish with undulate edge. Young
colonies were perfectly round, finely granular, brownish discs,
with sharply defined edge.

Strain LI. — Macroscopically ; like Ps. alba. Microsa >pically :
round, entire, yellow-brown, finely granular, concentrically banded
or zoned. The edge was comparativclv thin.

Ps. longa, and Ps. mesenterica. — Macroscopically; like Ps.
alba. Microscopically; round, brownish discs, somewhat zoned:
internally granular becoming nucleated at the center; the per-
iphery was thinner, coarsely granular, and zoned with granular
edge. A thin bordering zone showed coiled or curled surface
markings. The colonies of the two strains at first were alike,
but in 4 days slow liquefaction began in those of Ps. mesenterica.
10. Agar colonics. — Ps. alba. Growth rapid. Colonies
were at first round to irregular, smooth, edge thin and entire.

M [Cro i irg \\ i-Ms of M \ri.i: S \i' 569

Microscopically; edge thin, undulate, finely granular (motile or
ganism) ; grumose inwards center. Deep colonies yellov* brown
and grumose or coarsely granular. Later, macroscopically, the
colonies were undulate spreading; microscopically, brown and
grumose, the edge ragged, consisting of chains of organisms
extending into the medium. Spreading colonies were numerous.
(See Plates XV and XVI).

Ps. fluoresceins. — Colonies at first punctiform becoming
round, irregular and ameboid, characteristically surrounded by a
hazy, ill defined, outer zone. Spiny, filamentous processes often
frinsred the colonies. The internal structure was at first finely
granular, becoming coarsely granular to grumose at the center
with a finely granular outer zone. Curled, interwoven filamenl
were frequently seen in the interior. Medium strongly fluorescent.

Ps. longa. — Macroscopically ; punctiform, round, becoming
irregular or ameboid, spreading, raised and smooth; edge at first
entire, later more or less undulate. Microscopically; thin and
finely granular, the edge entire to undulate. The internal struc-
ture varied from finely to coarsely granular to grumose, floco i
or curled.

Ps. mesenterial. — The colonies of this strain could not be
distinguished from those of Ps. longa except that they were fre-
quently thinner and more widely spreading.

Ps. tenuis. — Punctiform becoming round or irregular.
Microscopically; finely to coarsely granular with edge entire to
broken and irregular, brownish and usually surrounded by a
thin transparent granular zone.

Ps. putrida. — Punctiform to round and irregular, edge entire
to broken. Colonies raised, convex, smooth to slightly con
voluted. Microscopically; thin, finely granular to grumose, and
showing concentric zones with radiate to irregular surface mark-
ings; the edge often becoming slightly undulate.

Strain CXII. — Colonies were round to ameboid and fre-
quently thin and spreading. Surface smooth, sometimes concen
trically ringed; usually raised, frequently becoming effuse to

57o Bulletin 167

hyaline. The edge varied from entire to undulate, frequently
thin and spreading. Internal structure, finely to coarsely granular
to grumose, often filamentous or tioccose ; commonly nucleated.
Medium strongly fluorescent.

Strain CXV. — Punctiform to round and irregular, typically
becoming ameboid spreading, frequently nucleated, somewhat
raised to convex, becoming effuse or hyalin. The ti\ge was en-
tire to undulate or broken. Buried colonies irregular with spiny
processes. Medium not fluorescent.

Strain CXL. — Round to irregular, raised, smooth, entire to
broadly undulate, sometimes broken. Microscopically; finely to
coarsely granular, grumose towards the center and commonly
showing irregular lines, being more or less floccose or curled.

Strain CXLV. — Round, raised to convex, smooth with the
edge entire to curled. Microscopically; finely to coarsely gran-
ular, becoming grumose ; frequently floccose or curled.

Strain CXLJ'III. — Macroscopically ; round, smooth, raised.
with edge thin, entire to undulate. Microscopically; finely to
coarsely granular or grumose, filamentous and curled to floccose.
Medium strongly fluorescent.

Strain XXX I 'I. — Macroscopically; round, smooth, raised,
entire to thin and undulate, frequently becoming effuse. Micro-
scopically ; finely to coarsely granular, grumose, filamentous and
curled to floccose; sometimes the interwoven chains of cells sug-
gested an anthrax colony. Colonies were often surrounded by
a thin transparent zone with an undulate edge.

Strain LI. — Macroscopically; round, irregular to ameboid,
smooth, raised with edge entire to undulate. Microscopically;
nucleated, finely to coarsely granular, becoming grumose towards
the center.

11. Cohn's nutrient solution (27:197). — This medium was
prepared as follows :

Distilled water 1000. CC.

Di-potassium phosphate 5. gr.

Magnesium sulphate 5.

Neutral ammonium tail rale 10.

Potassium chlorid 0.5

Micro-organisms of Maple Sap 571

Jt was heated to dissolve the salts, filtered and autoclaved.
Growth upon this medium occurred only with Ps. alba, Ps. longa,
and strain LI (also strain LIV identical with strain LI. c. E. page
550). After 20 days, transfers of those failing to develop were
made to broth and good typical growth resulted. The develop
ment of the three strains upon this medium was accompanied by
more or less blue-green to yellow-green fluorescence. Multiple
twinned crystals of magnesium ammonium phosphate formed
at the surface, which upon attaining sufficient size (about 1 cm.
in length), sank to the bottom where they were imbedded in a
viscid sediment.

The medium was varied by substituting mono-potassium
phosphate for di-potassium phosphate without significant effect
upon the development. The character of the growth was the
same upon half strength Cohn's solution.

Cultures developed a moderate clouding with white flaky
particles in suspension and a white, membranous pellicle hear-
ing crystals on the surface. More or less blue-green fluores-
cence appeared in the upper portions of the medium. Strain IJ
(LIV) and Ps. longa developed somewhat more slowly than Ps.
alba and often showed a scum rather than a membranous pellicle.
Typically twinned crystals were constantly associated with
development. Similar crystals were artificially formed by ex-
posing sterile tubes of Cohn's solution in a sealed jar with very
dilute ammonia. Liter flasks of Cohn's solution were inoculated
with strains LI and LIV and a sufficient quantity of the crystals
obtained for chemical analysis. The results showed that they
were magnesium ammonium phosphate due to the ammonia pro-
duced by the bacteria.

12. Uschinsky solution (27:197). — The media were pre-
pared as follows :

Distilled water 1 000. <•<•.

Glycerin (Merck's Blue Label) 40. gr.

Sodium chlorid ' •

Calcium chlorid 1-

Magnesium sulphate 0.4

Di-potassium phosphate - 1

Ammonium lactate 7.

Sodium asparaginase 4.

572 Bulletin 167

This was filtered and divided into two portions, one of which
was used full strength and the other diluted with an equal volume
of water. They were autoclaved, and inoculated at the same time
from the same stock cultures.

The results attained are summarized in the following- table :

TABLE 63. USCHINSKY SOLUTION

Normal solution Diluted solution

Strain Fluo- Fluo-

Growtli Time rescence Time Growth Time rescence Time

Ps. alba,

+

2d.

+

2d.

+

Id.

+

Id.

Ps. fluorescens,

+

2d.

+

2d.

+

2d.

+

2d.

Ps. longa,

+

2d.

-+-

2d.

+

Id.

—

10 d.

Ps. mesenterica,

+

2d.

+

2d.

+

Id.

+

Id.

Ps. tenuis.

+

2d.

±2

2 (1.

+

2d.

+

2d.

Ps. putrida,

—

10 d.

—

10 d.

—

CXI I,

+

2d.

+

2d.

+

Id.

+

Id.

CXV,

—

10 d.

—

10 d.

—

CXL,

+

2d.

10 d.

+

Id.

+

Id.

CXLV,

+

2d.

+

2d.

+

Id.

+

Id.

CXLVIII,

+

2d.

+

2d.

+

Id.

+

Id.

XXXVI,

+

2d.

+

Id.

+

Id.

LI,

+

10 d.

+

10 d.

+

Id.

+

I'd.

Modified Uschinsky solution was also prepared as follows :

Distilled water 1000. cc.

Glycerin (Merck's Blue Label) 30. gr.

Sodium chlorid 6. "

Calcium chlorid 0.1 "

Magnesium sulphate (tested purity) 0.4 "

Di-potassium phosphate 2. "

Ammonium lactate 6. "

Sodium asparaginate 4. "

This was filtered and divided into two portions, one of which

was used full strength, wrhile the other was diluted with an equal

\<>lume of water. Yet another solution was prepared leaving

out the ammonium lactate. This was likewise divided into two

portions, one being used at full concentration, the other at half

concentration. The four media were inoculated at the same time

from the same cultures.

Micro-organisms of M.\n.r, Sai?
The results are tabulated as follows:

TABLE G4. MODIFIED I S< KINSKY SOLI TION

Uschinsky

Half strength Uschinsky

Strain

Fluo-

Fluo-

Growth

Time

rescence

Growth

Time

rescence

Ps. alba.

+

20 hrs.

+

+

20 hrs.

+

Ps. fluorescens,

+

2d.

+

+

20 hrs.

+

Ps. Tonga,

+

20 hrs.

+

+

20 hrs.

—

Ps. mesenterica,

+

2d.

+

+

20 hrs.

+

Ps. tenuis,

+

20 hrs.

+

+

20 hrs.

+

Ps. putrida,

—

16 d.

—

—

14 d.

—

CXII,

+

20 hrs.

+

+

20 hrs.

+

CXV,

16 d.

—

—

14 d.

—

CXL,

+

20 hrs.

+

+

20 hrs.

+

CXLV,

+

20 hrs.

+

+

20 hrs.

+

CXLVII1,

+

20 hrs.

+

+

20 hrs.

+

XXXVI,

+

2d.

+

+

20 hrs.

—

LI,

+

2d.

+

+

20 hrs.

+

Uschinsky, no lactate

Half strength, no

lactate

Ps. alba,

+

20 hrs.

+

+

20 hrs.

+

Ps. fluorescens,

+

20 hrs.

+

+

20 hrs.

+

Ps. longa.

+

20 hrs.

—

+

20 hrs.

+

Ps. mesenterica,

+

20 hrs.

+

+

20 hrs.

+

Ps. tenuis.

+

20 hrs.

+

+

20 hrs.

4-

Ps. putrida,

—

14 d.

—

—

14 d.

—

CXII,

+

20 hrs.

+

+

20 hrs.

+

CXV,

—

*14 d.

—

+

l14d.

—

CXL,,

+

20 hrs.

+

+

14 d.

+

CXLV,

+

20 hrs.

+

+

14 d.

+

cxlviii,

+

20 hrs.

+

+

14 d.

+

XXXVI,

+

20 hrs.

—

+

14 d.

+

LI,

+

20 hrs.

+

+

14 d.

+

1 Trace 14 days.

The development of Ps. fluorescens, Ps. mesenterica.
XXXVI, and LI was more rapid but less persistent on the di-
luted media than on the full concentrations. All strains except
Ps. longa, Ps. putrida, and CXV developed well with surface
membrane and fluorescence. Ps. longa grew well but without
fluorescence; no growth occurred with Ps. putrida or CXV.
Transfers from tubes of these latter strain- to nutrient broth
developed typically, showing that the lack of growth on the
Uschinsky solutions was not due to a failure to inoculate.

574 Bulletin i6y

13. Nitrogen. — A nitrogen free medium was made as fol-
lows (27:198) :

Water 500. cc.

Cane sugar 2.5 gr.

Mono-potassium phosphate 1.

Magnesium sulphate 0.5

Sodium chlorid 0.2

This was heated until dissolved, and divided into two equal
portions. To one was added 2 grams of ammonium tartrate, to
the other 2 grams of Bausch and Lomb's peptone.

The results upon these media are tabulated as follows :

TABLE 65. NITROGEN REQUIREMENTS

Tartrate solution Peptone solution

Strain Growth Time Growth Time

/•.v. alba, + ' ,L

Ps. fluorescens, + Id. Id.

Ps. longa, — + 1 d.

Ps. mesenterica, + Id. Id.

Ps. tenuis. + Id. Id.

Ps. jmtrida: + 1 d. 3 d.

CXII, + 3d. + Id.

CXV, + 3 <1. + 1 d.

CXL, + 3d. + Id.

CXLV, + Ld. + Id.

CXLVIII, + 3 d. 1 d.

XXXVI, + Id. + 1 d.

LI, + Id. + Id.

1 Ps. putrida showed the hest growth of any at three days, with
beautiful green fluorescence.

In order further to test the nitrogen requirements, the Fol-
lowing medium was made, considerable care being taken to make

it nitrogen free.

Twice distilled water 3000. cc.

Sucrose 15. gr.

Mono-potassium phosphate (tested purity, no

nitrogen ) 6.

Magnesium sulphate (tested purity, no nitro-
gen) 0.3

Sodium chlorid (tested purity) 1.5

After being thoroughly mixed this solution was divided into

ten equal portions. One of these was reserved for a control, to

another was added 2'/, of peptone, while to each of the other

eight portions was added respectively o.S'r of the following

Micro-organisms of Maple; Sap

.. , .1

chemicals: asparagin, sodium asparaginate, urea, potassium
nitrate, calcium nitrate, ammonium lactate, ammonium phosphate,

ammonium tartrate. The ten media were inoculated at the same
time from the same cultures.

TABLE G6. NITROGEN REQUIREMENTS

Strain

Ps. alba.

Ps. fluorescens,

Ps. longa,

Ps. mrsent erica.

Ps. tenuis

Ps. putrida,

CXII,

CXV,

CXL,

CXLV,

CXLVIII,

XXXVI,

LI,

Ps. alba.

Ps. fluorescens,

Ps. longa,

Ps. mesenterica,

Ps. tenuis.

Ps. putrida,

CXII,

CXV,

CXL,

CXLV,

CXLVIII,

XXXVI,

LI,

Aspar-
agin

G. F.

+
+
+

+
+
+

Sodium
aspara-
ginate

G. F.

+
+
+
+

+

Urea
G. F.

+

Potassium
Peptone nitrate

+ +

+ + + +

+
+
+
+
+

+
+

+
+

+ +
- +

+ +

G.

+
+
+
+
+
+
+
+
+
+
+
+
+

F. G. F.

+

+

+ + +

+
+
+
+
+

Calcium
nitrate

G. F.

Ammo-
nium
lactate

G.

+
+
+
+
+

+

F.

+
+

+

Ammo-
nium
phosphate

C. F.

Ammo-
nium
tart rate

G. F.

+ +

+
+

+
+

+
+
+

+
+
+
+
+

Note.— G., growth; F., fluorescence; +, observed; — , not observed.

Observations upon these media were continued for over a
month. Peptone was the only source of nitrogen used by all
strains. Except with strain CXV, good growth was apparent in
24 hours; the latter showed good growth in 3 days. All strains,
except Ps. putrida and CXV, were able to utilize asparagin and

576 Bulletin 167

sodium asparaginate. Upon ammonium lactate the results were
practically the same; however, with Ps. longa and LI, growth was
somewhat delayed. Ps. alba, Ps. longa, Ps. mcsenterica, Ps.
tenuis, XXXVI, and LI, used ammonium tartrate as a source of
nitrogen. Upon the ammonium phosphate medium Ps. mcsen-
terica, Ps. tenuis, and XXXVI, developed. With potassium
nitrate, strain CXII showed a delayed growth. Xone of the
strains developed on the calcium nitrate medium. Ps. mcsen-
terica was the only strain which developed upon the medium con-
taining urea.

III. Physical and Biochemical Features.

For the purpose of determining acid production triplicate
titrations of cultures and controls were made after 1, 2, 4, 10, and
20 day incubation periods. Five cc. of the medium were pipetted
into 45 cc. of distilled water in Erlenmeyer flasks, boiled two
minutes and titrated hot with N/20 sodium hydroxid against
phenolphthalein.

1. Sugar free medium. — Control titrations were made upon
a sugar free medium prepared as follows : 1% of Bausch & Lomb's
peptone was added to distilled water and steamed in a double
cooker about 50 minutes, tubed 10 cc. per tube, and autoclaved.
A small amount of acid was produced on this sugar free medium
by strains Ps. putrida, CXII, CXV, CXLVIII. Previous to boil-
ing the acid was masked by ammonia. The figures in the table
indicate the percent of normal acid or alkali produced.

Plate XV. — Types of agar colonies. Figure 1. Ps. alba (two day old
colony showing anthrax-like strands). Figure 2. Ps.
strain CXLVIII (two day old colony). (See pages 568-570.)

'

Plate XVI. — Types of agar colonies. Ps. fluoresceins var. non-liquifa-
■ iens, strain CXL; two day old colonies, showing grnmose struc-
ture. (See pages 568-570.) X 40.

Micro-organisms of Maple Sap

--.77

TAItLE 67. PERCENT NORMAL A.CID OH ALKALI PRODUCED IN SIOAR

FREE MEDIUM

1

day

2

days

4

days

10 days

20 (

lays

Strain

Acid

Alk.

Acid Alk.

Acid Alk.

Acid

Alk.

Acid

Alk.

Ps, alba,

0.09

0.21

0.14

0.36

0.24

Ps. jiuurescens,

0.05

0.13

0.15

0.15

0.02

Ps. Tonga,

0.09

0.15

0.10

0.29

0.27

Ps. mesenter

ica,

0.1G

0.14

0.20

0.31

0.28

Ps. tenuis.

0.12

0.10

0.18

0.25

0.24

Ps. patrida,

0.04

0.12

0.06

0.09

0.53

CXI I,

0.09

0.00

0.00

0.05

0.14

0.22

CXV,

0.01

0.06

0.04

0.02

0.34

CXL,

0.0G

0.12

0.06

0.29

0.23

CXLV,

0.05

0.10

0.18

0.34

0.19

CXLVIII,

0.04

0.06

0.03

0.16

0.47

XXXVI,

0.06

0.07

0.18

0.24

0.28

LI,

0.02

0.07

0.10

0.25

0.20

2. Sugar media. — To i% peptone solutions identical with
the control, 2% of dextrose, lactose, sucrose, and glycerin respec-
tively were added. The several media were treated like the
control except that they were sterilized by the intermittent method.
Analytical results obtained with these four media are shown in
tabular form. In general, previous to boiling, the acid produced
was masked by the ammonia formed.

TABLE 68.

PERCENT NORMAL

ACID

OR ALKALI

PRODUCED ON

DEXTROSE

PEPTONE

1

day

2

days

4 i

lays

10 days

20 days

Strain

Acid

Alk.

Acid Alk.

Acid

1 Alk.

Acid

Alk.

Acid

Alk.

Ps. alba,

0.02

0.06

0.08

0.06

0.11

Ps. fluor.,

0.03

0.15

0.14

0.08

0.09

Ps. longa,

0.02

0.03

0.03

0.03

0.04

Ps. mes.,

0.12

0.14

0.26

0.29

0.36

Ps. tenuis,

0.12

0.12

0.14

0.31

0.67

Ps. putrida,

0.00

0.00

0.03

0.00

0.00

0.03

0.00

0.00

CXI I,

0.21

0.30

0.35

0.38

0.49

CXV,

0.00

0.00

0.09

0.00

0.00

0.02

0.00

0.00

CXL,

0.00

0.00

0.01

0.13

0.25

0.28

CXLV,

0.05

0.08

0.24

0.07

0.16

CXLVIII,

0.05

0.16

0.30

0.44

0.52

XXXVI,

0.11

0.07

0.10

0.00

0.00

0.00

0.00

LI,

0.02

0.02

0.05

0.00

0.00

0.00

0.00

578

Bt'I.T.KTIX 167

PERCENT NORMAL ACID OR ALKALI PRODUCED ON LACTOSE PEPTONE

1

day

2 days

*>
O

days

10 days

20 1

lays

Strain

Acid

Alk.

Acid Alk.

Acid Alk.

Acid

Alk.

Acid

Alk.

Ps. alba.

0.10

0.21

0.23

0.23

0.15

Ps. Jiuor..

0.1G

0.24

0.20

0.04

0.19

Ps. longa,

0.00

0.00

0.05

0.14

0.20

0.17

Ps. mes.,

0.10

0.13

0.16

0.21

0.22

Ps. tenuis,

0.06

0.14

0.13

0.15

0.15

Ps. putrida,

0.07

0.04

0.08

0.03

0.46

CXII,

0.08

0.0G

0.11

0.25

0.41

CXV,

0.02

0.05

0.06

0.05

0.36

CXL,

0.08

0.16

0.12

0.12

0.09

CXLV,

0.11

0.15

0.15

0.2]

0.15

CXLVIII.

0.02

0.02

0.15

0.41

0.93

XXXVI.

0.10

0.13

0.09

0.09

0.04

LI,

0.00

0.00

0.07

0.11

0.11

0.01

PERCENT NORMAL ACID OR ALKALI PRODUCED ON SUCROSE PEPTONE

1

day

2

days

4

days

10 days

20

days

Strain

Acid

Alk.

Acid Alk.

Acid Alk.

Acid

Alk.

Acid

Alk.

Ps. alba,

0.03

0.19

0.12

0.15

0.25

Ps. jiuor..

0.05

0.05

0.16

0.09

0.08

Ps. longa,

0.02

0.00

0.00

0.09

0.10

0.17

Ps. mes.,

0.03

II. IIS

0.12

0.14

(1.17

Ps. tenuis.

0.00

0.00

0.05

0.09

0.07

0.15

Ps putrida,

0.02

0.00

0.00

0.11

0.35

0.11

CXII.

0.0G

0.20

0.24

0.65

0.56

CXV,

0.06

0.05

0.00

0.00

0.08

0.10

CXL.

0.01

0.03

0.09

0.11

0.17

CXLV,

0.00

0.00

0.03

0.12

0.14

0.16

CXLVIII,

0.00

0.00

0.00

0.00

0.15

0.58

0.60

XXXVI.

0.03

0.01

0.15

0.02

0.01

LI,

0.04

0.06

0.14

0.09

0.14

PERCENT NORMAL A< 111 oi: ALKALI PRODUCED OX GLTCERIN PEPTONE

1

day

2

days

4

days

10 days

20

(lays

Strain

Acid

Alk.

Acid Alk.

Acid Alk.

Acid

Alk.

Acid

Alk.

Ps. alba,

0.12

0.19

0.21

0.24

0.19

Ps. fluorescens,

0.07

0.13

0.06

0.08

0.36

Ps. longa.

0.05

0.13

0.14

0.28

0.22

Ps. mesenterica,

0.07

0.15

0.17

0.24

0.05

Ps. tenuis.

0.01

0.12

0.16

0.13

0.11

Ps. putrida,

0.02

0.04

0.02

0.22

0.71

CXII.

0.10

0.03

0.02

0.12

0.26

CXV,

II. (Ml

11. nil

0.03

0.03

0.07

0.20

CXL,

0.04

0.17

0.19

o.is

0.16

CXLV.

0.11

0.16

0.12

0.2G

0.16

CXLVIII.

ii.ni

0.03

0.13

0.69

0.87

XXXVI,

0.04

11.14

0.02

0.13

0.29

LI,

0.05

0.1 1

0.08

0.22

0.1 1

M [CRO-ORGANISMS OF MAPLE SAP

3. . Iction on nitrates in nitrate broth. — Medium :

Water 1 ■ cc.

Peptone ( Witte's) L. gr.

Potassium nitrate, c. p -•

The cultures were tested for nitrites by the iodo-starch reaction

as follows [i~ :<>$). A starch paste was made by boiling a 4' ',

aqueous mixture of potato starch; to the 10 cc. of culture ! cc.

of the starch paste and 1 cc. of freshly prepared potasium iodid

water (0.4' < 1 were added, followed by three drops of sulphuric

acid (2 parts c. p. sulphuric acid to 1 part water). One series

of cultures and controls was tested upon the second, fifth, and

ninth da) s.

A diphenylamine reagent was prepared as follow- (32:340) :
5 grams of diphenylamine were dissolved in 100 cc. of pure con-
centrated sulphuric acid and added to 20 cc. of water, hi case
of negative nitrite tests with the iodo-starch reaction, a few drops
of diphenylamine reagent were poured down the side of the tube
in such a manner as to form 2 layers. A blue contact zone in-
dicated the presence of nitrates. When a positive nitrite reaction
was observed the nitrate test was performed with another cul-
ture.

Second day. — A positive nitrite reaction (immediate black-
ening) was given by Ps. mesenteriea, XXXVI, and Id. Nitrates
were present in all by the diphenylamine reaction. Ammonia
was indicated in all except CXV by Nessler's reagent.

Fifth day. — An immediate reaction for nitrites was given b)
Ps. mesenteriea. XXXVI, and LI. Nitrates and ammonia were
indicated in all.

Ninth day. — Immediate positive reaction for nitrites occurred
in cultures of Ps. mesenteriea. XXXVI, and LI and Ps. fluo-
rescens showed traces. Nitrates were indicated in all. A posi-
tive reaction for ammonia was obtained from all culture-, espe-
cially strong in Ps. putrida and CXLVIII.

A nitrate medium in which tap water was substituted for
distilled water was employed in fermentation tube-. The tests
were conducted as before, about 10 cc. of the medium being
poured into clean test tubes, for this purpose.

580 Bulletin 167

Sixth day. — Immediate nitrite reaction occurred in the fol-
lowing: Ps. mcscnterica, XXXVI, LI and a trace in Ps. fluo-
resceins, while nitrates were found in all. No gas was observed.
A test for nitrites was also made by the naphthlamine-sulphanilic
acid reagent. A positive reaction was obtained with Ps. mesetv-
terica, XXXVI, LI, and Ps. fluorcsccns. In the latter the color
was not as deep as in the others.

Eleventh day. — Only slight clouding in the open arm was
observed ; no gas was produced. A positive nitrite reaction was
obtained with Ps. fluorcsccns, Ps. mcscnterica, XXXVI, and LI.
Nitrates and ammonia were present in all by the diphenylamine
and Nessler's reagents respectively. A strong reaction with
Nessler's reagent was given by Ps. putrida, Ps. fluorcsccns, CXII,
and CXLVIII.

4. Indol. — The tests for indol on nutrient broth and on
Dunham's peptone solution were made by adding 10 drops of
chemically pure sulphuric acid and 1 cc. of .02% sodium nitrite to
10 cc. of culture. The tubes were allowed to stand 10 minutes
and then heated. Strains Ps. alba and CXL gave a trace of a
reaction in 10 minutes without heating. The results on nutrient
broth are given in table 69 and those on Dunham's peptone
solution in table 70. A positive result is indicated by +, a
negative by — , and a trace by ±.

TABLE G9. IXDOL PRODUCTION IX NUTRIENT BROTH.

Strain 10 13 18 20 22 24 26 30 30 30 37 40 40 40 60

(1. d. d. d. d. d. d. d. d. d. d. d. d. d. d.

/'.v. alba, + + + + + — + ± + — ±± + +

Ps. fluor.. — — — — — — — — — — — ± — — —

Ps. longa, ■ — — ■ — ± — — ± ± ± ± — — — — +

Ps. vies., — — — ■ — — — — — — — ■ — — ■ — ±

Ps. tenuis. — — — — — — — — — ■ — — — ±

Ps. putrida, — — — — — — — ■ — — ■ — — — — — —

CXII, _______ — — _ — ±

CXV, ____ — _______ — _ —

CXL, + + + + + + + + + + ± + + + +

CXLV, ___ — _ — — ±

CXLVIII, — ±

XXXVI, — ± — — ± — —

LI, — — — — — ±±±±± — ±

Micro-organisms oi? Maple Sap 58]

TABLE 70. lM'ol. PRODUCTION IN DUNHAM'S PEPTONE

Strain. 2 days G days 11 days 26 d

Ps. alba, ± +

Ps. fluoresce us. ±

Ps. longa, — — ±

Ps. mesenterica, — —

Ps. tenuis, — — •

Ps. putrida, — — — —

CXII,

CXV, ± ±

CXL, + +

CXLV,

CXLVIII, t

XXXVI,
LI, — • — —

5. Toleration of acids and alkalies. — Three liters of nutrient
broth were prepared in the usual way and divided into 13 por-
tions. These separate lots were adjusted, by titrating hot against
phenolphthalein with N/20 sodium hydroxid and N/20 hydro-
chloric acid, to the following reactions: — 37-5; — 30.0; — 23.8:
— 13.5;— 11. o;— 7.3:0.0; +8.8; +14.6; +25.0; +34.0 Fuller's
scale.

Since alkaline media rapidly change in reaction owing to
carbon dioxid absorption, control tubes of the several . media
were subjected to the same conditions as the cultures and their
reactions determined after 1 and 3 days. Table 71 indicates the
results secured upon these alkali and acid media after 24 hours'
incubation. The original reactions of the several media and, also,
the reactions after 24 hours are given at the top of the table.
Table 72 indicates the reactions of the media and the growth of
the organisms after a 3 days' incubation.

582

r.ii.i.KTix i <<j

TABLE 71. (JKOWTH ON ACID AM) ALKALI MEDIA AFTEB 24 HOURS

Strain
Ps. alba.
Ps. fluorescens,
Ps. Tonga.
Ps. vie sent erica,
Ps. tenuis.
Ps. putrida,
CXII,
CXV,
CXL,
CXLV,
CXLVIII.
XXXVI,
LI,

Reaction of media previous to tubing
0.0 —7.3

Reaction after

+0.13 —2.0

+ +

(Fuller's Scale)
-11.0 -13.5 —23.8 37.0

24 hours. (Fuller's scale)

— 4.9 — 7.4 —14.2 —17.9

+
+
+

+
+

+
+

+

+
+
+

+
+

+

Reaction of media previous to tubing (Fuller's Scale i

Strain

Ps. alba,

Ps. fluorescens.

Ps. longa,

Ps. mesenterica,

Ps. tenuis.

Ps. putrida,

CXII,

CXV,

CXL,

CXLV,

CXLVIII,

XXXVI,

LI,

-37.5

Reaction

-22.1

+8.8

after

+ 8.7

+
+
+
+
+
+
+

+
+
+
+
+

•.'1

+ 14.6

hours.

+ 12.:,

+

+
+
+
+

+
+
+
+
+

+ 25.0 +34.0

( Fuller's scale)

+ 22.0 +33.7

+ , growth: — , no growth; ±, doubtful growth.

Micro-organisms oe Maple Sap

583

l\r.li: 72. GROWTH ON A t ■ 1 1 > AND ALKALI MEDIA AFTEB 3 DAYS

Reaction after

Strain

Ps

Ps
Ps
Ps.
Ps
Ps

alba,

fluorescens,
longa,
mesenterica,

tenuis.
putrida,
CXI I,

cxv,

CXL,

CXLV,

CXLVIII,

XXXVI,

LI,

+3.0

+
+
+
+
+
+
+

+
+
+
+
+

+0.4

+
+
+
+
+
+
+

+
+

+

days. t ( Fuller's scale)
—2.2 —3.3 —10.0

+ -

1 l.C

+

+

Strain

Ps. nlbit.

Ps. fluorescens,

Ps. longa.

Ps. mesenterica.

Ps. tenuis.

Ps. putrida.

CXII,

CXV,

CXL,

CXLV,

CXLVIII.

XXXVI,

LI,

Reaction after :! days. (Fuller's scale)

—20.0

+10.9

+
+
+
+
+
+
+

+
+
+
+
+

13.5

+ 24.1

+

+

+

+

+

—

+

+

+

—

+

—

+

+

+

—

+

—

+

+

+

+

+

+

+

—

• 34.G

+, growth; — , no growth; ±, doubtful growth.

6. Optimum reaction for grozi'th in bouillon. — In connec-
tion with the experiments on the toleration of sodium hydroxid
and hydrochloric acid, observations were made after a day's in-
cuhation as to the optimum reaction for growth. The best
growth with all strains was found to occur in a very slightly acid
medium. The figures given in table y$ represent the original
reaction of the media in which growth occurred in 24 hours
( c. f. page 582 for actual reactions). The reactions at which
the best growth occurred are shown in black face.

584 Bulletin 167

TABLE 73. OPTIMUM REACTION

Strain
Ps. alba,

Ps. fluorescens, — 7.3

Ps. longa,
Ps. mesenterica,
Ps. tenuis,

Ps. putrida, — 11.0 — 7.3

CXII,
CXV,1
CXL,

CXLV, — 7.32

CXLVIII,
XXXVI,
LI,

Reactions

0.0

+8.8

+ 14.(5

0.0

+8.S

+14.6

0.0

+8.8

+14.0

0.0

+8.8

+14.(1

0.0

+8.8

+14.(5

0.0

+8.8

+14.(5

0.0

+8.8

+14.(5

0.0

+ 8.8

0.0

+8.8

+14.G

0.0

+8.8

+14.(5

0.0

+8.8

+14.(5

0.0

+8.8

+ 14.(5

1 No growth at 24 hours.
8 Trace of growth.

7. Vitality on culture media. — Long vitality on culture
media was characteristic. Five month agar cultures completely
dried down, developed promptly with fluorescence when trans-
ferred to nutrient broth.

8. Temperature relations; thermal death point. — Thin walled
tubes of uniform thickness with an inside diameter of 15 to 17
ram. were chosen for the determinations of the thermal death
point. Seven cc. of bouillon, -f-10 Fuller's scale, were employed
in each case. The inoculations were made from young broth
cultures, care being taken not to wet the tube above the surface
of the medium. In the preliminary determinations the tubes
were deeply submerged in the thermal bath for 10 minutes at the
following temperatures: 44.5, 47.7, 54.5, 58.4, 61. 30 C. After
exposure the tubes were incubated at 25 ° C. In this way the
thermal death point for all strains was roughly fixed between
47.7 and 54.40 C. None grew after exposure at 54.5 ° C.

In subsequent trials the temperature of the bath was varied
so as to determine the death point more closely. A very large

Micro-organisms of" Maple Sap

585

number of tests were made in an effort to fix the exact death
point accurately, but the experiments indicate clearly that the
death point of these organisms under the conditions" employed,
is not constant within at least 0.5 ° C. The final conclusions are
recorded in the table below :

TABLE 74. THERMAL DEATH

POIN r

Strain

°C.

°C.

Ps. alba,

between

53.5 and 53.9

Ps. fluorescens,

a

53.1 '

' 53.5

Ps. putrida,

u

53.5 '

1 53.9

Ps. longa,

CI

51.1 '

* 51.5

CXL,

II

51.5 '

' 51.8

CXLVIII,

tt

51.5 '

' 52.9

CXLV,

(t

50.2 '

' 50.5

CXII,

it

50.2 '

' 51.5

CXV,

it

50.2 '

' 50.5

XXXVI,

It

50.2 '

' 50.5

Ps. mesenterica,

it

49.3 '

' 50.2

LI,

1"

49.1 '

' 49.3

Ps. tenuis,

It

48.3 '

' 49.1

Optimum temperature. — -The best growth with all strains oc-
curred at approximately 25 ° C.

Maximum temperature. — At 36.7 to 35 ° C. only Ps. fluores-
cens and Ps. putrida developed in 24 hours. At 33.4 to 30.80
in the same length of time Ps. alba. Ps. fluorescens. Pr. longa,
Ps. mesenterica, Ps. putrida, CXII, and CXL, showed growth.
At 31.2 to 28.50 Ps. albo, Ps. fluorescens, Ps. longa, Ps. mesen-
terica, Ps. tenuis, Ps. putrida, CXII. CXL, and LI developed.
The experiments indicated that all strains could grow at tem-
peratures up to 330 C. Only I's. fluorescens, Ps. putrida, and
CXV developed at 360 C. in 7 days. In no case was the growth
at these higher temperatures as good as it was below 300 C.

9. Drying. — Small cover slips were placed on bits of pa-
per in petri dishes and sterilized in the oven. Each was then
inoculated with one loop of young broth culture, the age of
which varied in the different experiments from 1 to 3 days. They
were allowed to remain in the dark at room temperature. At
intervals, cover slips were removed and dropped into tubes of

586

Bulletin [67

sterile broth. A characteristic growth after incubation was re-
garded as evidence of continued vitality. It is to be noted that
in general the organisms retained their vitality longer when
taken from the older cultures than when take from day old

cultures.

TABLE

10. re;

3ISTAP

rcE TO

DESIC<

•ATION

Strain

Days

dried

1

2

2

2

3

q

0

3

4

4

Ps. alba,

+

—

+

+

—

+

+

Ps. fluorescens,

+

+

+

+

Ps. Ion (in.

+

+

—

—

Ps. mesenterica,

+

+

—

Ps. tenuis.

—

Ps. putrida,

+

+

+

+

+

+

+

+

CXII,

+

—

+

+

+

+

+

+

+

cxv.

—

+

T

—

CXL,

+

+

+

—

—

CXLV.

+

+

+

-t-

+

+

+

CXLVIII.

+

+

+

+

+

+

+

+

XXXVI,

+

+

+

+

+

+

+

+

4.

1

LI,

+

+

+

—

Strain

Days

dried

6

6

6

10

14

IS

20

30

17

Ps. alba.

—

—

Ps. fluorescens.

—

—

—

Ps. longa,

—

—

—

Ps. mesenterica,

—

Ps. temii.s.

—

Ps. putrida.

+

+

+

+

+

CXII.

+

+

+

+

—

+

—

—

—

CXV,

—

— .

—

CXL,

+

CXLV,

—

—

-

CXLVIII,

+

+

+

+

+

—

XXXVI.

—

+

+

—

+

—

LI.

+

+

—

+, growth: — , no growth: ±, doubtful growth.

10. Insolation. — Agar plates, one-half covered were ex-
posed on snow to bright sunlight for 10 minutes at n a. m.
Feb. 29. All were sensitive, the percent killed varying from
J^ to IOO%. The number of colonies on the unexposed half.

A I [CRO-ORG VNISMS OF M Al-I.i- SAP

587

the number on the exposed half, and the percent killed are in-
dicated below :

TABLE "li. INFLUENCE <>K INSOLATION

Strain

Number of colonies Number of colonies
on unexposed half on exposed half

Ps. all>a.

22:.

70

Ps. fluorescens,

340

100

Ps. longa,

G8

21

Ps mesenterica*

24

2

Ps. tenuis,

280

18

Ps. putrida,

numerous

0

CXII,

18

0

cxv,

280

5C

CXL,

numerous

0

CXLV,

186

142

cxlviii;

8

2

XXXVI,1

countless

42

LI,

numerous

0

Percent
killed

69
To
GO
92
93

too

lint
80

inn
23
7.".
95

||H>

1 Percent could only be estimated on account of spreaders.

Ten minutes exposure of strains CXII. CXV, CXL,
CXLV, CXLVIII, XXXVI, and LI, March 8, at n a. m. gave
results indicated in table 77.

TABLE 77.

INFLUENCE OF INSOLATION

Number of

Number of

Plate

colonies on

colonies on

Percent

Strain

No.

unexposed half

exposed half

killed

CXII,

1

350

0

too

2

113

0

LOO

CXV,

1

38

0

Kin

2

G

0

100

CXL,

1

1494

II

100

2

2289

0

100

CXLV,

1

381

0

1(11)

2

540

II

LOO

CXLVIII,

1
2

1

318

127

(id

5,

540

226

58

■2

763

85

89

XXXIII.

1

countless

i)

ion

2

it

0

KID

XXXVI,

1
2
1

381

I)

inn "

LI,

L58

(I

L00

2

222

1

99 5

588 ButtETIN 167

11. Acids produced.- — Hydrogen sulphid. Tubes of nu-
trient broth were prepared and inoculated in the ordinary way
and in each, including controls, was suspended a strip of filter
paper thoroughly moistened with lead acetate solution. Blacken-
ing of the paper after a suitable incubation of the cultures in-
dicated the presence of hydrogen sulphid. The following strains
gave this reaction constantly, CXV, CXL, Ps. tenuis. With
strain CXV it was in evidence in 2 days; with Ps. tenuis and
CXL, in 5 to 6 days. A slight reaction was observed in tubes
of LI and a doubtful trace occasionally in Ps. putrida, CXII,
and CXLV.

12. Alkalies produced. — Ammonia was indicated by Nes-
sler's reagent in nitrate broth cultures of all strains (c. f. page

579)-

13. Crystals. — Crystals of magnesium ammonium phos-
phate were formed in Cohn's solution by Ps. alba, Ps. longa and
strain LI.

14. Diastasic action on potato starch. — Broth cultures of
the 13 strains were tested for diastasic action upon potato starch
after incubation periods of varying duration. The results were
negative.

15. Anaerobiosis. — Pyrogallic acid oxygen absorption meth-
od : Freshly prepared agar slants were placed in a liter Novy
jar containing 15 grams of pyrogallic acid. Just before seal-
ing about 200 cc. of a 1% sodium hydroxid solution was added.
After 10 days incubation in the sealed jars a slight growth
with fluorescence was apparent in strains Ps. alba, Ps. flito-
rescens, Ps. longa, Ps. mesenteric a, Ps. tenuis, CXII, CXL.
CXLV, CXLVIII, XXXVI, and LI. No growth resulted
with Ps. putrida, or CXV ; however, normal growth occurred
in tubes of the latter two strains soon after they were removed
from the jar.

Carbon dioxid method. — Carbon dioxid was produced from
washed and boiled marble and hydrochloric acid in a Kipp gen-
erator and purified by passing successively through solutions of

Micro-organisms of" Maple Sap 589

sodium carbonate, potassium permanganate, pyrogallic acid in
i'/o sodium hydroxid, and distilled water and allowed t<> pass
for 2 hours through a Novy jar containing freshly prepared agar
slants. The jar was sealed and held at room temperature. No
strain showed growth after 5 days incubation, but in all cases
good growth occurred in from 1 to 2 days after the seal was
broken. A second series incubated for 10 days gave duplicate
results. In addition to the plain agar slants, lactose and dex-
trose agar slants were used with similar results. The reaction
of the incubated agar medium was roughly determined by tritur-
ating 5 cc. of the solid medium in 45 cc. of distilled water and
titrating. Sufficient carbon dioxid had been absorbed to give
the cold medium a reaction of +20 Fuller's scale.

In order to be sure that the failure to grow in the carbon
dioxid was not due to the acidity of the medium, augmented by
absorbed carbon dioxid, the following anaerobic methods were
employed.

Hydrogen method. — A series of plain agar slants and a
series of 1% dextrose litmus agar slants were placed in a Now
jar and the air displaced by hydrogen. The jar was exhausted
in order to hasten the displacement. The litmus agar remained
neutral, and titrations of the plain agar controls without boiling
demonstrated that the reaction had not been changed. No
growth occurred in 10 days. All developed well when removed
from the jar.

Roux method. — Sterilized Roux tubes were aseptically filled
with young broth cultures of the 13 strains. This test was tried
several times and in no case did growth appear except in de-
fective tubes or in those containing air bubbles. After incuba-
tion of 10 days the tubes were broken and the contents gathered
in sterile test tubes where normal growth resulted.

These tests show that the various strains are strictly aerobic.
They further indicate that lactose and dextrose are not fer
mented in the absence of oxygen. (Consult papers of Andrewes
and Ilordu (1) and Glenn (to) in bibliography).

590

I'.n.i.KTTX 167

GROUP NUMBEE OF T1IK 13 STRAINS

The group number of the 13 strains was easily computed
with the exception of the digits representing the action of the
bacteria upon carbohydrates and glycerin. On account of the
formation in the control, i. e., peptone solution, of decomposition
products having an acid reaction, there is some doubt in certain
cases whether the acid in the sugar and glycerin peptone media
resulted from oxidation of the introduced compounds, or from
proteid decomposition. In each case the amount of acid pro-
duced in the carbohydrate media was compared with the amount,
if any, produced in the control, and where the difference was
less than 0.2% the digit representing no acid production was
recorded. However a comparison of the tables will show that
no sharp line of differentiation exists between acid production
and no acid production.

The group numbers determined were as follows:

Ps. alba,
Ps. longa,
Ps. putrida,

Ps. mesenterica,
Ps. LI,
Ps. tenuis,
Ps. CXL,

Ps. fluoreseens,
Ps. CXII,
Ps. CXLV,
Ps. CXLVIII,
Ps. XXXVI,

B. CXV,

212.2332133
212.3332133
212.3332132

91 1 9000-100
Z ± J. . i- o o o 1 o o

211.2233133
211.2322133
211.2222133

211.2223132
211.2222133
211.2332133
211.2222132
211,2223132

211.3332?33 Doubtfully fluorescent

Non-1 iquetiers

Tardy liquefiers

Rapid liquefiers

species

As already noted bacteriological literature contains descrip-
tions of over 50 species of bacteria which are capable of pro-
ducing green fluorescence upon the common media. However.
it is probable that many so-called species are identical with.
or varieties of, previously named species. The following refer-
ences from literature are cited as illustrations of the trend of
i ipinion.

M [CR0-0RGAN ISMS 01? M M'l.i. SAP

.V.I I

Niederkorn (23) concluded that there are only two constant
forms, B. fluorescent liquefaciens Fliigge and B pyocyaneus
Gessard, among the following:

pyocyaneus,

a

Gessard.

tt

b

Ernst.

tt

c

Preudenreich.

a

pericardii .

Harold-Ernst.

it

void bakt. Institut. Travel

tt

strumit. Lanz, Bern.

fluoresceins

liquefaciens

Fliigge.

tt

alb us

Adametz.

tt

a 11 reus

Zimmermann.

a

Ion gus

tt

tt

tenuis

(«

a

mesenteric us

Tataron".

tt

putridus

Fliigge.

a

capsulatus

Pottien.

a

liquefaciens

(vom Vert", mis Wasser i

Ruzicka (24) shows that B. pyocyaneus and B. fluorescens
liquefaciens vary so widely within the species and so overlap in
their cultural characters that no sharp line of differentiation be-
tween them can be drawn.

Griffon (11) states that B. caulivorous, B. brassicaevorous
and B. aeruginosas are not to be considered as distinct species,
but only as forms of B. fluorescens which, under a favorable en-
vironment, easily changes from a saprophytic to a parasitic ex-
istence. He holds that B. aeruginosas is to be considered
synonymous with B. fluorescens putridus.

According to various authors: B. fluorescens non-lique-
faciens is synonymous with B. fluorescens putridus and repre-
sentative of the group comprising Bacillus fluorescens tenuis.
Bacillus fluorescens aureus and Bacillus fluorescens erassus;
Bacillus viscosus Frankland is synonymous with Bacillus fluores-
cens liquefaciens, and Bacillus fluorescens fuscus corresponds to
Bacillus oogenes fluorescens.

In view of the above citations it is evident that the fluo-
rescent organisms are closely related, probably being varieties of
one polymorphic species. Before stating the conclusions which
have been drawn concerning the number of species represented
in the seven strains of sap bacteria and the six known -train-

•'»''♦- Bulletin 167

which were studied, attention is directed to a few clauses ab-
stracted from Fliigge's description of his Bacillus fluorescens
liquefaciens (6:11: 292). "The degree of liquefaction varied
very considerably. There were varieties which in 2 days and
others which first in 2 weeks liquefied the gelatin ; the form of
the colonies varied, also, from spherical to spreading with bor-
der entire to toothed. The optimum temperature was 200 to
25 ° C. Many varieties do not grow at 37 ° C, others grow well
and form on agar a more or less thick layer. The oxygen re-
quirements are equally variable ; through them are explained the
different forms of liquefaction in stab cultures. If one wishes
to conceive all minor variations as constant characters, one must
make dozens of varieties."

According to Fliigge (1. c.) Bad. bittyri fluorescens La-
far, B. fluorescens nivalis Schmelck, Bacillus viscosus Frank-
land and B. fluorescens minutissinius I una may be regarded as
synonymous with B. fluorescens liquefaciens. In the description
of B. fluorescens non-liquefaciens, he says. "A sharp line between
the liquefaciens and non-liquefaciens variety of B. fluorescens
does not exist since there are forms which in the first day of plate
growth appear to belong to the latter group, which later sink
gradually in the gelatin."

It is believed that the morphological, cultural, physical and
biochemical features of the 13 strains of green fluorescent bac-
teria warrant the assumption that there are but two species rep-
resented: Pseudonwnas fluorescens and an unknown species,
strain CXV, which, as has been previously noted (c. f. page 546),
never evidenced more than doubtful fluorescence.

Ps. fluorescens is represented by 12 strains which are di-
visible into two varieties, liquefaciens and non-liquefaciens. The
following strains belong to the liquefaciens variety: fluorescens
inescntcrica, CXII, CXLV, CXLVIII, XXXVI and LI. To the
non-liquefaciens variety belong, alba, longa, putrida and, pro-
visionally, tenuis and strain CXL, which showed a very tardy

Micro-organisms os M\iu: Sap

liquefaction of gelatin but in other respects closely resembles
the other three strains.

New species. — Strain CXV : This strain appears to be dif-
ferent from any of the others studied. A feeble trace of fluor-
escence was thought to have been present at times in agar cul-
tures. Repeated staining for flagella has shown that the latter
are peritrichiate. Another peculiarity about this organism i-
the parallel grouping of single or double cells common in cap-
sule and flagella preparations, c. f. Plate X, figures 3 and 4. As
previously noted several cells commonly occurred side by side in
the same capsule with several flagella arising about the periphery
of the latter. A careful consideration of the character of tlii-
bacillus seems to justify the conclusion that it is a distinct specie--.
The name Bacillus parallelus is proposed for this organism. For
purposes of reference the differential characters of tlii- new
species are summarized as follows:

594 Bulletin i6y

BRIEF DESCRIPTION OF BACILLUS PARALLELUS

( NEW SPECIES )

I. Morphological Characters

i. Form. — A short motile bacillus with rounded end- oc-
curring singly, typically in twos; long chains common upon
solid media.

2. Size. — Individual cells had the following dimensions.
( )n hanging: blocks: length 1 to 2.6 microns, diameter .8 to 1.0
microns. Hanging drops; length [.6 to 4.0 microns, diameter
.5 to .<j micron, stained preparations; length .8 to 2. microns,
diameter .5 to .8 microns.

3. Grouping. — Short and long chains; delicate membran-
ous pellicle.

4. Bndospores. — None observed.

5. The flagella were peritrichiate : tufts were common at
each end of short chains, but lateral flagella were also present
on what appeared to be individual cells. Easily demonstrated
by Ldwit's or Loeffler's method.

6. Capsules. — Capsules easily demonstrated upon prepara-
tions from 10 day broth cultures by Welch's method or Richard
Muir's contrast stain. Frequently 2 and as many as 5 cells oc-
curred side by side within the same capsule with flagella arising
about the periphery of the envelope.

7. Zoogloea. — Small zoogloea masses were common in
liquid cultures.

8. Staining reactions. — Gram's stain: Deeply stained even
after 15 minutes in absolute alcohol; with arnyl alcohol. Gram's
stain was irregularly retained after 5 minutes' exposure. Aqueous
fuchsin: deeply stained. Aqueous gentian violet: well stained.
Carbol fuchsin ( Ziehl ) : well stained.

II. Cultural Features
1. Agar stroke. — Not characteristic; a narrow filiform.
white slim) to viscous band. Doubtful fluorescence of the
medium.

Micro-organisms of Maple Sap 59.f

.'.»..

_, Potato. — A nanow. slimy, filiform band, becoming
brownish; sometimes the growth became dull and more or l<
rhizoid in character; the medium was more or less grayed.

3. Agar stab. — Best growth at top, colony at first white,
becoming brownish; development somewhat restricted; the line
of puncture was at first beaded to filiform, becoming somewhat
villous. The medium showed a very slight fluorescence and in
old cultures became amber brown.

4. Gelatin stab. — Growth best at the top; line of puncture
filiform to beaded; liquefaction at first crateriform, later strati-
form, beginning in 24 hours and proceeding rapidly at first : com-
plete in 62 days.

5. Nutrient broth. — Rather slow growth ; moderate uniform
clouding in from 2 to ; days; transient; white membranous
coherent precipitate; old cultures were brownish and stringy.

6. Milk. — Digestion apparent in about 4 days; a firm co-
agulum appeared in about 18 days accompanied by acid produc-
tion; digestion was completed in from 30 to 104 days : the medium
never appeared green but was more or less straw colored
throughout.

7. Litmus milk. — Alkaline reaction in 4 days, succeeded by
slight acid production when digestion began; the litmus was com-
pletely reduced in 2 or 3 weeks' time.

8. Gelatin colony. — Rapid growth : first punctiform to round
and entire, rapidly sinking in the saucer-like depressions of the
liquefying gelatin.

9. Agar colonies. — Punctiform and round to irregular and
ameboid; the colonies effuse or raised and convex; the edge thin
entire to undulate and broken; finely to coarsely granular to
grttmose; commonly nucleated. Colonies dense, dark brown,
fringed with spiny processes occurred les> frequently.

10. CoJin's solution. — Xo growth.
it. Uschinsky solution. — Xo growth.

12. Nitrogen requirements. — Nitrogen obtained from pep-
tone and ammonium tartrate.

596 Bulletin 167

III. Physical and Biochemical Features

1. Action upon carbohydrates in peptone solution. — Neither
acid nor gas were formed in 1% peptone solutions containing
2% of either dextrose, lactose, sucrose or glycerin.

2. Ammojiia production. — Moderate ammonia production;
18.6 cc. N/10 hydrochloric acid equivalent produced in 100 cc. of
broth in 10 days.

3. Nitrates in nitrate broth. — Not reduced. Nitrates and
ammonia present ; ammonia probably from proteid decomposition.

4. Indol production. — Doubtful in 11 day Dunham's pep-
tone cultures after boiling ; absent in nutrient broth.

5. Toleration of acids and alkalies. — Slight.

6. Optimum reaction for growth. + 15 Fuller's scale.

7. Vitality on culture media. — Long.

8. Temperature relations. — Thermal death point; between
50.2 and 50.50 C. Optimum temperature approximately 250 C. ;
maximum, 37 ° C ; minimum, 5-70 C.

9. Resistance to desiccation. — Continued vitality after 3
days drying on cover slips ; 4 days fatal.

10. Insolation. — Sensitive ; 80 to 100% killed after 10
minutes' exposure.

11. Acids produced. — Abundant hydrogen sulphid produc-
tion.

12. Alkalies. — Moderate ammonia production.

13. Relation to oxygen. — Strict aerobe.

14. Group number. — Bacillus 21 1.3332 ?33.

BRIEF CHARACTERIZATION OE THE 12 STRAINS OE Ps. fluOreSCeUS

For the convenience of any one who may have occasion to
review these results the brief characterization items of the six
strains of fluorescent bacteria isolated from maple sap together
with those of strains alba, fiuorcscens, longa, mesenterica, tenuis,
and puirida are summarized in tabular form on pages 598 anil
599-

Micro-organisms of Maple Sap 591

Conclusions

Among the 42 strains of green fluorescent bacteria selected
from several hundred strains which had been isolated from
maple sap, there were 32 strains of the liquefaciens and 9 strains
of the non-liquefaciens varieties of Ps. fluorescens. The studies
included one strain which was never more than doubtfully fluo-
rescent which, for this and other reasons, should not properly be
regarded as a member of the fluorescent group. The 9 strains
of the non-liquefaciens variety showed a delayed liquefaction of
gelatin in from 50 days to 5 months when cultivated in a moist
chamber at 200 C. Peptonization of milk by these strains was
also long delayed.commencing in from 30 days to 3 months.

Critical comparative studies of 7 representative strains of
green fluorescent sap bacteria and 6 so-called species Ps. alba.
Ps. fluorescens, Ps. longa, Ps. mesenterica, Ps. tenuis, and Ps.
putrida show that no sharp line of differentiation can be drawn
between these forms. Of the known "species,"' Ps. alba, Ps.
longa, and Ps. putrida fail to liquefy gelatin in 6 months time.
while in the case of Ps. mesenterica and Ps. tenuis a delayed
liquefaction occurs. In the latter named strain liquefaction first
appears 4 months after inoculation.

It is believed that the fluorescent sap bacteria as well as
the so called species, Ps. alba, J's. longa, Ps. mesenterica, Ps.
tenuis, and Ps. putrida should properly be recognized as -trains
of the liquefaciens and non-liquefaciens varieties of Ps. fluores-
cens.

;98

BUT.T.KTIX Ihj

TABLE 78. BRIEF CHARACTERIZATION

/.

a

-

'—

<
w

Diameter over 1 micron

Chains

Endospores

Capsules

Pseudozoogloea

Motile

Gram's stain

Cloudy . .
Pellicle .
Sediment

Agar
Gel. plate

Shining
Round .

Surface-growth
Needle-growth. .

o

■-
<

w

'—

Potato

Moderate
Discolored

Grows, at 37° C

Grows, in Conn's solution...
Grows, in Usehinsky solution

OJ o

= r

— —

Gelatin *

Casein

Agar

i

Coagulated '-'

Casein peptonized ::

Indol
Hydrog
Amnion
Nitrates
Flu ores

ia

3 reduced

cent

1 Under observation (> months.

-Coagulum more or less jelly-like in consistency.

'Under observation 4 months.

Micro-organisms of Maple Sap

590

01 i in I- strains of Ps. fluorescens.

a

-a

+

+

+

—

+

—

+

+

+

+

+

+ ' +

+

+

+

+ +

+

1— <

e

—

__'

>

>

>

<~

4:

a

HH

-

-

]

y.

X

S

*

"a

X

X,

X

X

y.

—

~~

•~

-

Q

c

b

X

c

*

w

s

1i

■*~

a

d

=

d

c

^

>— -

^

•Ki

a

-

eS

-

■a

.

.

<

%-

£h

—

i—>

-r

JZ

■r

■r.

<

^_

4-1

-i->

—

+j

fin

a.

Bh

^

Os

73

73

w

03

73 1

— -
73

+

+

+

+

+

+

+

+

+

+

+

+

+

•>

•>

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

1

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

i-

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

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+

+

+

+

+

+

+

+

+

+

4- + + + +

+

+
+

+

+

+
+

+

+

+

+

+
+

+

+

+

+
+

+

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+

-+■

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+

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-+-

+ +

+

+

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600 I'.ui.i.htin 167

Bibliography (Part III C)

1. Andrewes, F. W. and Hordu, F. J. A study of the Streptococci

pathogenic for man. Lancet II, p. 708 (1906). Streptococci
refused to attack lactose under ordinary conditions, but did so
readily under anaerobic conditions.

2. Belli, C. M. Chemische, mikroscopische und bakteriologische Un-

tersuchungen iiber den Hagel. Centralb. f. Bakt. VIII Bd. 2
Abt., pp. 445-446 (1902).

3. Chester, F. D. A Manual of Determinative Bacteriology (1901).

4. Edson, H. A. Buddy sap. Vt. Sta. Bui. 151 (1910).

5. Ellis, D. Outlines of Bacteriology, p. 108 (1908).
1;. Fliigge, C. Die Mikroorganismen (1886).

7. Fred, E. B. Uber die Beschleunigung der Lebenstatigkeit hoherer

und niederer Pflanzen durch kleine Giftmengen. Centralb. f.
Bakt. XXXI Bd. 2 AM., pp. 185-245, 198-202 (1911). The
author shows the effect of various dilutions of ether, carbon
disulphid, "potassium dichromate, copper sulphate, and salvar-
san (606) upon B. fiuorescens liquefaciens and B. pyocyaneus.

8. Gessard, C. Des races du bacille pyocyanique. Ann. de l'lnst.

Pasteur, V, pp. 65-78 (1891). Not seen.

9. . Sur la fonction fluorescigene des microbes. Ann.

de l'lnst. Pasteur, VI, pp. S01-823 (1892). Not seen.

10 Glenn, T. H. Variation and carbohydrate metabolism of bacilli of

the Proteus group. Centralb. f. Bakt. CLVIII Bd. 1 Abt., p.
481 (1911). P. vulgaris, though not able to ferment lactose
under aerobic conditions, does so readily under anaerobic con-
ditions.

11 Griffon, E. Sur le role des bacilles fluorescents de Fliigge en

pathologie vegetale. Compt. rend. Ac. Science Paris, No. 1, 149,
pp. 51-53 (1909). Reviewed in Exp. Sta. Red, XXVI, 3, pp. 246
(1911). Original not seen.

12. Harrison, F. C. Bacterial content of hailstones. Botanical Ga-

zette XXVI. pp. 211-214 (1898).

13. Jensen O. Bakteriologische Studien iiber Danische butter. Cen-

tralb. f. Bakt. XXIX Bd. 2 Abt., pp. 610-616 (1911).

14. Jordan, E. O. The production of fluorescent pigment by bacteria.

Botanical Gazette, XXVII, pp. 19-36 (1899).

Micro-organisms of Maple Sap 001

L5. . Bacillus pyocyaneus and its pigments Jour. \'.\

per. Med. IV. 5-G, pp. C27-G47 I L899).

16. Laurent, Emile. Recherches experimentales sur les malades des

plants. Ann. de l'lnst. Pasteur, XIII, pp. 1-4S (1899). Not
seen.

17. Lepoutre, L. Recherches sur la transformation experimental*' de

bacteries banales en races parasites des plants. Ann. de
l'lnst. Pasteur, XVI, p. 304 (1902)). Not seen. Reviewed,
Centralb. f. Bakt. X Bd. 2 AM., pp. 189-190 (1903).

is. Luxwolda, W. B. "Wachstum unci Wirkung einiger Milchbakterien
bei verschiedenen Temperaturen. Centralb. f. Bakt. XXXI Bd.
2 Abt, pp. 129-175 (1911). B. fluorescens liquefaciens firsl
coagulates milk with a lab ferment and then peptonizes it.
The author gives the numbers of bacteria present and the de-
grees of acid in milk cultures after various incubation periods
at different temperatures. He says that B. fluorescens lique-
faciens develops remarkably fast at low temperatures, 3°-5° C.

19. Maassen, A. Die Zersetzung der Nitrate und der Nitrite durch Bak-

terien. Centralb. f. Bakt. VIII Bd. 2 AM., pp. 152-154 (1902).

20. Marchal, Emile. The production of ammonia in the soil by mi-

crobes. Centralb. f. Bakt. I Bd. 2 Abt., pp. 753-758 (1895).

21. Migula, W. System der Bakterien (1900).

22. Muir, R. and Ritchie, J. Manual of Bacteriology (1905).

2.'.. Niederkorn, E. Vergleichende Untersuchung uber die verschie-
denen Varietaten des Bacillus 'pyocyaneus und des Bacillus
fluorescens liquefaciens. Centralb. f. Bakt. XXVII Bd. 1 Abt.,
pp. 749-750 (1900).

24. Ruzicka, S. Experimentelle Studien uber die Variabilitat wich-

tiger Charaktere des B. pyocyaneus and des B. fluoresci ns
liquefaciens. Centralb. f. Bakt. XXIV Bd., pp. 11-17 (1898).

25. Schmelck, L. Steigerung des Bakteriengehalts im Wasser wahrend

des Schneeschmelzens. Central!), f. Bakt. IV Bd., pp. 195-199
(1888).

26. . EJine Gletcherbakterie. Centralb. f. Bakt. IV Bd.,

pp. 545-547 (1888).

27. Smith, E. F. Bacteria in relation to plant diseases, I. (1905).

28. . II, (1911).

602 I'.n.i.KTix 167

29. Smith, E. F., Brown, N. and Townsend, C. 0. Crown-gall of

plants; its cause and remedy. U. S. Dept. Agr., Bu. PI. Ind.,
Bui. 231 (1911).

30. Thoni, J. Biologische Studien fiber Limonaden. Centralb. f. Bakt.

XXIX Bd. 2 Abt., pp. G16-G43 (1911).

:', 1 Thumm. Karl. Beitrage zur Biologie der fluorescierenden Bak-
terien. (Inaug. Diss.) Arb. a. d. Bakter. Inst. d. Techn, Hoch-
schule zu Karlsruhe I Heft. 2, p. 291 (1S95).

32. Treadwell, F. P. and Hall, W. T. Analytical Chemistry, I, p. 340

( 1908).

::::. Wright. Jour. Boston Society Med. Sci. •",, p. 114 (19(H)).

34. Zimmermann. Die Bakterien unserer Trink-und Nutzwiisser, I.
Reihe (1890).

Im»i:\

INDEX.

Analysis of maple sirups from inoculated saps. .392-397, 102-412, 122 171

Analytical standards in maple sugar work 123, 163-472

Aseptic conditions, Bacterial content of sap obtained under.... 341

Ash content of maple sirup 163

Bacillus aceris, Analysis and discussion of sirups inoculated

with 142

Bacillus aceris, Capsule 182

Bacillus aceris. Cultural characters, mel hods Is 1

Bacillus aceris. Cultural features of 17C, 184

Bacillus aceris. Cultures on agar handling blocks 180

Bacillus aceris. Description of 175

Bacilli's aceris. Detailed description of * ~s

Bacillus aceris. Dimensions 480

Bacillus in eris, Fission 181

Bacillus aceris. Form 179

Bacillus aceris. Grouping Is'

Bacillus aceris. -Involution forms 183

Bacillus aceris, Morphology of IT-".. 180

Bacillus aceris. Motility and flagella Is 1

Bacillus aceris. Physical and biochemical features of 177. 194

Bacillus aceris, Spores 182

Bacillus aceris. Staining reactions 's:;

Bacillus aceris. Summary of characters of 175

Bacillus parallelus, Cultural features of 59 1

Bacillus parallelus, Description of 59 •

Bacillus parallelus. .Morphological characters of 59 1

Bacillus parallelus. Physical and biochemical features of 59 1

Bacterial content of sap obtained under aseptic conditions... . ::il

Bacterial content of sap obtained under septic conditions 341

Bacterial flora, Influence of container on 343

Biochemical and physical features of Bacillus parallelus 59 1

Biochemical and physical features of green fluorescein organ-
isms 532, 57G

Chart showing grouping of 142 strains of green fluorescent or-
ganisms 550

Chemical and physical data, Summary of 47::

Chemical and physical data as to inoculated sap sirups 419-474

Cocci of maple sap. Pink 516

Comparative studies of green fluorescent organisms 552

Composites, Analysis and discussion of sirups classed as 11"

604 Bulletin 167

Conclusions 41G

Conclusions concerning green fluorescent organisms 597

Container, Effect upon quality of sirup of the 372

Container on bacterial flora, Influence of 343

Contents, Table of r 323

Controls, Analysis and discussion of 421

Cultural features of Bacillus aceris 476, 484

Cultural features of Bacillus parallelus 594

Cultural features of green fluorescent organisms 528, 557

Depreciation value of sirups 413

Description of Bacillus parallelus 594

Experiments, General plan of field 343

Experiments in 1909, Inoculation 349-::.".'.'

Experiments in 1910, Inoculation 359-380

Experiments in 1911, Inoculation 380-390

Failures, Analysis and discussion of sirups classed as 431

Field experiments, General plan of 343

Field experiments, Statistical summary of 391

Fluorescent bacteria. Analysis and discussion of sirups inocu-
lated with and with spore-bearers 440

Fluorescent bacteria occurring in maple sap, Green 521

Fluorescent organisms, Conclusions concerning green 597

Gray yeasts, Analysis and discussion of sirups inoculated with. . 435

Green fluorescent bacteria occurring in maple sap 521

Green fluorescent organisms, Comparative studies of 552

Green fluorescent organisms, Conclusions concerning 597

Green fluorescent organisms, Cultural features of .528, 557

Green fluorescent organisms, Morphology of 527, 553

Green fluorescent organisms, Physical and biochemical features
of 532.

Green fluorescent organisms, Preliminary studies of 553

Green molds, Analysis and discussion of sirups inoculated with 448

Incubator controls. Analysis and discussion of 425

Inert extraneous material, Influence of 415

Inoculated and natural sour saps compared, Sirups from 112

Inoculation experiments in 1909 349-359

Inoculation experiments in 1910 359-380

Inoculation experiments in 1911 380-390

Insoluble ash content of maple sirup 463

Invert sugar content of maple sirup 461

Last run sap. sweet, Analysis and discussion of sirups classed as 444

I n i J-: x •'."..

Last run sap, Si nip from . . . I II".

Last run sour kept sap, Analysis aid discussion of sirup mad-'

from >^f 156

Last run, sour sap, Analysis and discussion of sirup made from 15 1

Malic acid values of maple sirup 163

Maple sap bacteria, Technical description of 17".

Micro-organisms in maple sap, Number of

Micro-organisms in sour maple sap, Number of

Micro-organisms in sweet maple sap, Number of

Micro-organisms to spoiled maple sap, Relation of

Morphological characters of Bacillus parallelus

Morphology of Bacillus aceris I

Morphology of pink cocci

Morphology of green fluorescent organisms ~<~l~, 553

Natural sour saps and inoculated saps compared, Syrups from.. 412

1909, Inoculation experiments in 3 19-359

1910, Inoculation experiments in 359-380

1911, Inoculation experiments in 380-390

Non-fluorescent bacteria, Analysis and discussion of sirups in-
oculated with 127

Occurrence in maple sap of green fluorescent bacteria 521

Physical and biochemical features of Bacillus aceris 177, 19 I

Physical and biochemical features of Bacillus parallelus 594

Physical and biochemical features of green fluorescent organ-
isms r, ::l\ 576

Physical and biochemical features of pink cocci 519

Physical and chemical data as to inoculated sap sirups 419-17 !

Physical and chemical data, Summary of 17'.

Pink cocci, Analysis and discussion of sirups inoculated with... 129

Pink cocci, Cultural characters 517

Pink cocci, Morphology 516

Pink cocci of maple sap 516

Pink yeast, Analysis and discussion of sirup inoculated with... 1 -"■ i!

Preliminary studies of green fluorescent organisms 553

Ps. fluorescens. Characterization of 12 strains of 598

Quality of sirup as affected by container 372

Red yeasts, Analysis and discussion of sirups inoculated with.. 133

Remedial measures 117

Scoring, Method of sirup '■' 15

Septic conditions, Bacterial content of sap obtained under :: 11

Sour last run sap, Analysis and discussion of sirup made from. 152

606 Bui.i.ktix 167

Soui' maple sap, Number of micro-organisms in 339

Sour sap. Sugar and sirup from 372

Sour saps compared, Sirups from inoculated and natural 412

Statistical data, Summary of 392-397,402-411

Statistical summary of field experiments 391

Summary of field experiments, Statistical 391

Summary of physical and chemical data 473

Summary of statistical data 392-397, 402-411

Sweet, last run sap, Analysis and discussion of sirups classed as 444

Sweet maple sap, Number of micro-organisms in 340

Sirup quality as affected by container 372

Sirup scoring, Method of 345

Sirups, Depreciation value of 413

Sirups from last run of sap 357, 379, ;js9, 415

Sirups made from inoculated and from natural sour saps com-
pared H2

Table of contents •••»••

■>_•!

Tap-hole infection, Influence of 342

Technical description of maple sap bacteria 475

Tin buckets vs. wooden buckets 372

Tin or in wooden buckets, Analysis and discussion of sirups

from saps collectea in 45Q

Total ash content of maple sirup pi-
Wooden or in tin buckets, Analysis and discussion of sirups from

saps collected in 45Q

Wooden vs. tin buckets 372

Yeasts. Analysis and discussion of sirups inoculated with gray.. 135

Yeasts, Analysis and discussion of sirups inoculated with red.. 433

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