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BACTERIA IN DEEP WELLS AND SURFACE WATERS

FRED WILBUR TANNER B. S. Wesleyan University, 1912

THESIS

Submitted in Partial Fulfillment

of the Requirements for the

Degree of

MASTER OF SCIENCE

IN CHEMISTRY

IN

THE GRADUATE SCHOOL OF THE

UNIVERSITY OF ILLINOIS

1914

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UNIVERSITY OF ILLINOIS

THE GRADUATE SCHOOL

dune 1, 1914 190

| HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY FRED WILBUR TANWER |

ENTITLED ...... BACTERIA IN DEEP WELLS AND SURFACE WATER.

BeeeaeGerieD AS FULFILLING. THIS PART OF THE REQUIREMENTS FOR THE

DEGREE OF . MASTER OF SCIENCE

In Charge of Major Work

WANG a Head of Department

Recommendation concurred in:

Committee

errant nr aeonyavaastl rcs ononsesscsensmns Se eee essen ban er Ditte : on

Final Examination

Part I (A) Bacteria in Deep DMD t ets Seemed aeadabdane 4 ade Part I (B)

Bacillus colon in Ground Waters.......ceseeoeeees

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Isolation of Bacterlacccccccccccccccccccccccccces Part II

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Of all living things, bacteria are most widely distributed over the surface of the earth. As is the case with higher animals, we find a definite habitat for a par- ticular species of bacteria. There are certain places where some can exist in greater numbers than can others. We some- times find species in an environment quite different from

the normal.

PART I (A) Bacteria in Deep Wells. Water coming from underground sources was, until recently, considered sterile. Many older text books claimed that there were no bacteria in water from underground sources. More recently, however, investigators have reported bacteria in deep well waters, until at the present time, most text books grant that a ground water may contain bacteria. EE reports from 6 to 26 cells per cca. in a Kent well sunk in chalk. He gives no information with regard to the methods used in securing the results. ieeiate’ found from 6 to 30 bacteria per cc. in some artesian wells at Kiel. The medium used is not given. Hueppe is reported to have found only four ina

deep well at Wiesbaden where a special investigation was

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carried on. In the Maing wells Egger found four colonies.

Savers” claims that deep well water having been filtered through layers of earth should contain few bacteria and should be subject to very little variation. On gelatin the number of bacteria is usually less than 50 per cc. and on agar less than l per cc.

Prescott and Winslow” quote analyses of deep wells and springs in the neighborhood of Boston in which the number of bacteria varied from 0 to 12. They reach the conclusion that water absolutely free from bacteria is not ordinarily secured from any source.

Thresh* gives many instances of bacteria in ground waters and especially of the intestinal flora coming under

his own observation. Some of these will be mentioned later.

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The results in the following table indicate the

number of bacteria found in some Illinois deep wells:

Sample No. Depth Agar Gelatin 27452 2 9 55 27474 400 0 0 27475 160 1 1 27476 160 28 30 27477 120 “= 6 27545 211 & 10 27557 895 0 20 27568 113 4 3 27582 2000 1 9 27611 126 0 2 27612 126 0 6 27672 270 s 10

EXPERIMENTAL

It was thot advisable to investigate the character of the colonies found on some of the plates of water from underground sources. These wells are located at different points in the state of Illinois and all are strictly speaking deep wells. Only those samples were considered which were known to be from wells which had been pumped for some time. Very often it was found that a badly contaminated deep well water came from a new well which had been thoroly flushed out.

The work here presented was done on the samples as

they came to the State Water Survey for the routine analysis.

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Method of Procedure. The water was plated on agar and incu- bated at 37°C and on gelatin at 20°C. After these plates had been counted, colonies were picked off by means of a sterile platinum wire and transferred to agar slants. These cultures were later plated, in dilution, on gelatin in order to secure a pure culture. The cultural characteristics were then determined by means of the different standard media . and the results recorded on the charts of the Society of American Bacteriologists.

Sample A.

A well 1382' deep located at Odell, Illinois,

furnished this water. It was comparatively new, but had

been pumped for some time before the sample was sent in. The well is cased with iron pipe and the cover is water tight. The water is securec from the St. Peter sandstone.

The number of bacteria on agar was 26 and on gel- atin, 1000. The high number of bacteria on gelatin may have been due to the fact that the casing had not been thor- oly flushed out when the sample was taken. The two follow- ing bacteria were taken from the gelatin ates and sub- jected to the different kinds of media mentioned below. The colonies were of the same general shape varying only in size. All grew on the surface and were colored slightly brown.at their centers. ‘The gelatin plates began to liquefy in less then forty-eight hours.

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a I. Bacillus subtilus.

Diameter: Less than 1 micron.

Spores: Formed toward middle of the rod.

Motility: Motile when taken from fresh broth mediun.

Gram: Positive.

Broth: Turbid with pellicle.

Gelatin plate: Gelatin is liquefied in about 40 hours. Thru the liquid granules occur.

Potato: Rich wrinkled growth.

Milk: Coagulated.

Indol: Negative.

Nitrates: Not reduced.

Gas: Negative.

2. Bacillus Flourescens liquefaciens.

Diameter: Less than 1 micron.

Spores: No spores could be found.

Motility: Positive.

Gram; Negative.

Broth: Sediment-broth assumes green color. Agar: Luxuriant growth - agar is turned green. Gelatin plates: Gelatin rapidly liquefied. Greenish color. Potato: Scant dark colored growth.

Milk: Coagulated and casein is digested. Indol: Slightly positive.

Nitrates: Ammonia is produced.

Gas; No gas is formed.

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Conclusions. Neither of these forms are of any sanitary Significance. Bacillus subtilis is a form abundant on grass. Bacillus floresceus liquefacieus is a common water form. Sample B

The source of this water was a 90' drilled well. The casing is sunk thru rock and clay and the water is pumped from the rock by an iron pump. Since the cover is of cement and watertight, no surface water can get into the well. The number of bacteria on agar was 7 and on gelatin, 67. The chemical analysis showed a normal water for such a source. The tests for Bacillus colon were negative. The colonies on gelatin were evenly distributed and had begun to

liquefy the gelatin in less than 43 hours.

I. Bacillus Vulgatus.

Diameter: Less than 1 micron.

Spores: Negative.

Motility: Positive.

Gram; Positive with pellicle.

Broth: Turbid with pellicle.

Agar: White.

Gelatin plates: Round liquefyers.

Potato: Seanty growth.

Milk: Coagulated. Partial digestion of casein. Indol: Positive.

Nitrates:

Gas: Negative.

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Conclusions. Bacillus vulgatus has no sanitary significance. It is a rather common forn.

Sample C

The source of this sample was a 2500' well. The

casing was put down thru rock and sandstone and had been

pumped for a long time. The count on agar was 30 and on

gelatin, 6. large liquefying colonies of a flourescent green

were present as in many of the deep well waters. ‘The colony

picked off had the following characteristic.

I. B. arborescens.

Diameter: Slender motile rod. Spores; Negative. “Motility: Positive. Gram: Negative. Broth: Agar; Yellowish. Gelatin plates: Rapid liquefyer - Dark opaque colonies with hairy projections. Potato; Orange colored growth. Milk: No change.

Indol: Negative. Nitrates: Reduced. Gas: No gas.

Conclusions. Bacillus arborescens is found

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Sample D

A well drilled in drift 1135' furnished this water.

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The well is cased with iron pipe and has a water tight cement cover. No feed lot, privy or stable is near the well. The gelatin plates were covered with liquifiers which made large saucer-like depressions in the medium. From this sample of

water the following organism was isolated.

i. Bacillus mycoides.

Diameter: Large bacillus.

Spores: Positive.

Motility: Positive.

Gram: Positive.

Broth: Cloudy with pellicle.

Agar: White growth irregular edge, after spreading. Gelatine plates: White with many branches.

Potato: White. Wo discoloration.

Indol: Negative.

Nitrates: Reduced to nitrites and armonia.

Gas: Negative.

Conclusions. Bacillus mycoides is a common species of bac- teria and might easily get into a water.

-Im all the above cases, it is realized that these different possibilities allowing bacteria to get into @ ground water. The well mizht be a new one which was not thoroly flushed out when the sample was taken. Or, it might have a bad casing allowing surface water to enter.

Since bacteria have been reported in deep wells

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from different parts of the world, it seems probable that

certain forms do live in deep well waters.

PART I. (B) Bacilius Colon in Ground Waters.

The presence of Bacillus colon in a water has been accepted by most sanitarians as a sufficient indication that the water has, in some way, received sewage pollution.

Bacillus colon was discovered in 1885 by Emmerich while working on the feces from cholera patients. Since it was found to be present in such large numbers, in that part of the intestine termed the colon, it was given the name Colon Bacillus. When it was first discovered, it was thot to be an inhabitant only of the human intestine. This theory, however, was very soon to be disproved. Flint? worked on the feces from the animals in the Chicago Zoological Garden. He found Bacillus colon in the excreta from the snake, llama, white rat, bear and a few others. He concluded that Bacillus colon was not @ sufficient basis on which to condemn a water. Belitzer® and Dyer and Keith “obtained re- sults to the same effect that Bacillus colon was not only present in the human intestine, but rather widely distributed thru the intestines of most warm blooded animals. Much other work has been reported by various men on Bacillus colon and its ubiquity in warm blooded animals.

Numerous instances are cited of its occurrence in

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ence of Bacillus colon in fish. He could not find it in twenty-three fish, including fourteen varieties. Johnson” examined sixty different fishes from the Illinois and Missis- sippi rivers. He succeeded in isolating Bacillus colon

from forty-seven. In forty-one of these the organism was isolated from the intestine. He cites the carrying of Bacillus colon by fish as a method by which a pure water céuld

be apparently polluted. As mentioned before, Flint proved

it in the snake. Moore and Wright’? could not find it in

the frog. Eyre®6 reports its presence in the fish and also in some warm blooded animals.

Prescott found an organism similar in all char- acteristics to Bacillus colon on grains from fields where animal contact was improbable. Even in this case will arise the possibility of birds distributing Bacilli Coli over such areas. One of Prescott's conclusions was that care should be used in interpreting an analysis of a water where Bacillus

colon was found to be present.

8

Metcalf reports Bacillus colon on some South

Carolina rice fields.

smith? found colon like bacteria on a field of rye in western Massachusetts.

Since colon organisms have been found in so many different places, even where animal contact was improbable, the question can be raised whether its presence can be taken

as an accurate indication of pollution. If it has as wide

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spread distribution as it seems to have, it is easy to imagine how it might gain access to a safe potable water. Prescott has indirectly proposed in one of his publications that this organism might originelly heave been a plant form, and finding the intestine of animals such a favorable abode, had taken up its habitation there.

ix {jn & paper in which no experimental

In 1894 Kruse date is given, advised against the use of this organism, as an indication of pollution. He said that we were dealing with a group of bacteria and not @ single organism. Since it was found in the air, water, and earth, he believed it could not be taken as a sufficient indication of pollution.

ces found Bacillus colon in the city supply of Strassburg. This water is taken from deep wells. In his work, he used large quantities.

arena? * after examining some deep and shallow wells about Parma concluded that B. colon had no sanitary significance.

WetasenZeld~ like Kruse stated Bacillus colon

could be found in all waters if large enough quantities

were taken. During his work, he studied about thirty sam-

ples of a supposedly good water.

On the other hana, we have those who contend that no good water should contain Bacilli coli. chick!* has this view. Savage” states that sufficient evidence has not yet

been produced to discard Bacillus colon as an indication of

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SA en ee eo eS | We

oe pollution. ‘the same statement has been made by Moore.

BACILLUS COLON IN DEEP WELLS

That we should find any indication of pollution in a ground water is queer, but since bacteria have such a wide distribution,it is possible. Many instances are given of contamination of under ground waters.

Horton?” in examining deep well and spring waters in Ohio often met with organisms resembling Bacillus colon. All shallow wells were excluded and only those wells which were cased, were considered. SBacillus colon was found twice at an interval of a month. The chemical data showed no pol- lution. Horton concluded that (1) Bacillus colon in ground water should condemn them and (2) the fact that water may come from underground sources should not be @ guarantee of its purity.

Nenkivel1° points out that water from wells in chalk are liable to intermittent pollution and should be puri- fied. Microorganisms may get into the water thru fissures and swallow holes from many miles distant, to infect an ap- parently pure water supply.

Thresh, in his book on water supplies, quotes many instances of finding Bacillus colon in deep well waters. He says, "There are few if any waters in which Bacillus colon

cannot be found if a sufficient quantity is taken."

The following work was done on a series of nineteen

tubular wells, constituting the supply for a city of about

hy 4

7

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saodd Elite dna bobu toxe boca ottew®

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fi

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set itqun <otae ne deee eee ot toot 9 ow qeeb at stoftod as ii bost gntiatt to 2058

1otea¢ et vilnas ?peberiigs & TL aniyo ad 3 to stiged & np'anot sev See agit wot Lot - natal vito & tot ‘feats o f2 gultst lt terds. J

-13-

eleven thousand inhabitants. The positive test for gas for- mation varied in the water, but were usually present in 1 cc. samples. ‘hese wells vary from 80' to 125' in depth. ‘They are all connected to the same pump suction so that it is impossible to secure a sample of water from any one well to see which one is furnishing the gas formers. Water is taken from the gravel deposits of a nearby river.

page No. 16 The following table/shows both the chemical and

bacterial analyses of the water from June 25, 1906 - May, 1914.

It shows a variation in quality which my be due to the intermittent entrance of another kind of water. The chlorine which is a valuable constituent by which to judge the qual- ity of a water varies between 61 and 35 p.p.m. Similar variations can be seen in the residue, oxyzen consumed and nitrogen content. ‘The number of bacteria is not excessive, but the almost constant presence of gas formers is a bad indication.

These wells are located near a large river carry-

ing a very highly polluted water. The drainage from the

- gurrounding country is towards the river, since the land

Slopes gradually away from it. No observations had ever been made to determine the direction in which the ground wa- ter moved. ‘The soil is of sand and gravel. There is no impervious stratum to protect the ground water from the sur-

face water which might get in.

Since the soil is so sandy, we may assume that

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there is more or less in filtration of surface water. We can hardly assume that the same standard of purity would be secured here as would be the case with a sand filter. There would not be a proper rate of filtration nor a satisfactory arrangement of sand and gravel to secure the highest effi- ciency. Old wells, fissures and cess pools might allow pol- luted water to get into the water bearing stratum with insuf- ficient purification.

_ There is a possibility of some underground connec- tions with the badly polluted river. ‘The following table has been prepared to show the relation of the constituents of the river water, the city water and water from two wells and of about the same depth, located within a half a block

__ of the city wells.

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River City Wells Private Private Well Well No.l Noe

Laboratory No. 27414 27416 27261 27262 Date 4/20/114 4/20/'14 3/28/'14 3/28/'14 Turbidity 50 0 2 0

Color Odor Resi due Chlorine. Oxygen Consumed

Nitrogen as Free Ammonia 3.520 024 -000 -000

Alb. Ammonia Nitrates Nitrites Alkelinity

Bacteria per cc. Agar

Gelatin

Gas Formation 10 cece

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The analytical results show a decided difference between the river water and that taken from the wells. Pollution from this source is improbable, altho a small amount of river water might account for the variation in the city well water.

Gas forming bacteria have been present in 10 cc. samples of the water in over 90 per cent. of the analyses made. The 1 cc. samples are positive in nearly 50 per cent. of the samles analyzed. In the 0.1 cc. samples a much better showing is made with only 9 positive tests from 64. At six different times, gas formers were isolated and found to have characteristics agreeing with Bacillus colon. Very rarely were there any liquefying bacteria present, but flourescent colonies quite often appeared. Some of these proved to Bacillus flourescens liquifacieus and Bacillus

flourescens non-liquefacieus, as indicated below.

ISOLATION OF BACTERIA

The methods of isolation were those commonly used and recommended by the Society of American Bacteriologists. The samples of water sent to the State Water Survey are packed in ice. Some of the samples which were studied were taken by representatives of the State Water Survey, by which means, we hoped to eliminate the danger of contamination by having inexperienced men take the samples. When the sample was brought to the laboratory it was plated on litmus lactose ager, Plain agar and plain gelatin. The red colonies devel-

? s +

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A

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-19=

oping on litmus lactose agar were picked off and purified by the usual methods.

Confirmatory tests were made according to the chart of the Society of American Bacteriologists. In addition, Endo's medium and Russell's medium was used. Both of these special media gave reactions characteristic of Bacillus colon.

The following characterisitics were assumed to be typical of Bacillus colon:

Shape: Bacillus 2 - 3 microns X .5 micron.

Motility: Motile with flagella.

Spore: Negative.

Gram: Positive.

Gelatin colonies: Small, thin colonies. No liquefaction. Gelatin stab: Thin growth more vigorous at the surface. Agar stab: Very scanty growth.

Agar stant: Thick growth.

Litmus milk: jAcid and coagulation efter a few days. Indol: Present after 3 days at 37°C.

Potato: Brown growth.

Nitrate: Reduced.

Lactose: Fermented giving gas and acid.

Dextrose: Fermented giving gas and acid.

Laccharose: Fermented giving usually gas and acid.

The chart giving the characters of the gas former isolated from the deep wells, is attached. Characters were not always constant. The motility varied somewhat, but gas

formation was constant. The organism from the deep wells

if oF BIL eoe SO OKer ceo poten moitiiba st seteteeioteerssd seats ‘to aa 6 (Foe bee Hue ‘fet hen eC Beemein Biz cfiouk te e423 atuetowtady co ROE arnig ats hacen eta ape eeraaha: testo ytro Lin? atte olde pars Show 3

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-20-

has characters identical with those of Bacillus colon. In this case the bacteriological analysis is more delicate than the sanitary chemical.

Attempt to Trace Source of Pollution.

Realizing that slight contamination was possible from these wells, salt was used as a means of trying to de- termine the source of pollution.

Different methods have been used to trace pollu- tion of underground waters; chief among these have been salt, lithium salts, flourescein and bacterial suspensions such as Bacillus prodigiosus.

Salt has been used in many instances for such pur-

oll used this method to demonstrate to the

poses. MacCollie citizens of Georgia the results which would be obtained, were a deep well used to dispose of sewage. It was found that a 124" drilled well would carry away an unlimited sup- ply of water and it was proposed to use this as a method of disposing of the sewage. A large amount of salt was put down the well and the chlorine content of the surrounding wells watched. The chlorine increased in the wells and springs in the vicinity, showing that there was underground connection with each well and spring. This demonstrated what would have resulted, had the well been used to carry

away the sewage.

Dolet® in 9 paper on the use of flourescein in

tracing water courses, comes to the following conclusions:

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(1) In studying the sanitary character of a well, it is more Valuable to study the underground flow then to analyze the water itself.

(2) Foreign substances put into an aquifer and traced from point to point, are of great value in this study.

(3) With the flourescope 1 pt. of flourescein in 10 billion pts. of water can be detected.

(4) Flourescein is a particularly valuable flow indicator for fissured and cavernized rocks.

(5) It progresses at a slightly slower rate than the water in which it is suspended.

(6) It is not decolorized by passage thru sand, gravel manure; it is slightly decomposed by calcareous soils.

(8) It is entirely decolorized by peaty formations and by free acids except by carbonic acid. These con- clusions give the limitations of this chemical in tracing ground waters.

M. Trillat?® has used many colored substances to trace motion of underground waters and claims that flour-

escein can be detected in dilutions of et a a ao e

He claims that before this dye is used Bivtrde Giaasien: a study of the soils should be made to determine the presence of any matters which might decompose the dye.

Marboutin, oO Gives an account of this dye when used to trace underground waters and comes to about the

Same conclusions that others have.

2 5 Martel ‘ shows that this dye even in very concen-

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trated solutions decolorizes rather quickly when kept in the sunlight. When it is kept in complete darkness, which would be the case in the earth, it did not change even after long periods of time.

dktenean” reports some work of Alba Orlandi and Roudelli, who used a suspension of Bacillus prodigrosus. They found that this organism found its way thru soil two hun- dred meters, when poured on the ground. In the same paper is quoted the work of Pfulil, who found that it took the same organism a short time to pass thru twenty-four feet of gravel. Gehrmann also reports an instance, coming under his own observation, where wells two to three hundred feet deep located too near an old canal were subject to entrance of contaminated water. No experimental data is given in this paper.

In the wells on which this work was done, it was thot best not to use flourescein on account of the possi- bility of coloring the water too much. Since these wells furnish the only supply for a city of9897 inhabitants, and Since it is difficult to remove this chemical effectively

under ordinary conditions, salt was used in an attemt to see if there was any seepage from the surface. |

One ton of fine salt was evenly divided between eleven privy vaults. This was placed in them on the same afternoon, the chlorine content of the water having been previously determined. The plot showing the location of

these privys with respect to the water works is given.

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When this experiment was sterted the chlorine content was 42 pts. per mil. From the table it will be seen that this changed scarcely at all.

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~24-

Date From To CC P.P.M. Remarks. March 16 22.5 26.7 4.2 42 No salt. = RT 22.5 26.9 4.4 44 Salt added. =) var 2h. 25.3 4.3 43 | 7 38 #£=2%.6 26. 4.4 ak ' 20 &2. 26.5 4.5 45 =. seCOBE. 25.3 4.3 43 > 39 #a1.? 26.1 4.4 oa 10 gals. water add- >. 88. 81.6 26.1 4.5 45 ed to all vaults. -— Be 2.6 26.2 4.4 44 =": Bt 22.8 27.2 $4.4 44 = os 81.5 26. 4.5 45 [ee 38.6 22.8 4.3 43 = 83 80,6 24.8 4.3 43 a. ae 19. 25.4 4.4 44 7 ae. 12%. 2525 4.3 43 = me 22, 26.2 4.2 42 | Rs 26.8 4.3 43 = 86 20.5 24.7 4.2 42 x 26 20. 24.2 4.2 42 % 27 21. 25.2 4.2 42 Heavy rein night of ee 6684S 28.8 4.3 43 3 ° * 28 21.5 25.8 4.3 43 " 28 20.5 24.7 4.2 42 Relea 84.6 25.8 4.3 43 Heavy rain night of a ae 2 25.2 4.2 42 3/29 > Ch 806 24.7 4.2 42 > Bi: 80.6 24.7 4.2 42 April 1 20.5 24.7 4.2 42 Heavy rain night of = 8 2. 25.2 4.2 42 3/31 a 3 2i. 25.2 4.2 42 = 4 22.5 26.8 4.3 43 b & 21.5 25.8 4.3 43 All day rein from by > 2&8. 26.2 4.2 42 6:00 A.M. ° G6 22. 26.2 4.2 42 ™ 9 21.5 25.7 4.2 42 Teh, 2. 2742 4.2 42 me “28. 26.3 4.3 43 Sots. BS. 272 4.2 42 ~~ ey SS 26.2 4.2 42 > 26 20. 24.2 4.2 42 7. 4 20,46 24.6 4.1 41 > “20. 82.8 26.6 4.1 41 Pumped for fire 2 hrs. > «ec 82.6 26.5 4. 40 Sample taken 45 min " 85 #22. 26.1 4.1 41 after fire out. — 86 280 24.1 4.1

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Since there was no increase in the chlorine con- tent, it is quite apparent that there is no direct connection

with pollution from surface sources.

PART II. Surface Waters.

At one of the large filtration plants on the Mississippi river two kinds of bacteria have been troublesome. Brief Description of Filter Plant. The plant is of

the rapid sand type furnishing about 4,000,000 gallons of water per day. The water is taken from the Mississippi river and has a normal chemical content for that water. The raw water is coagulated with alum, filtered and disinfected with caleium hypochlorite.

On the plates made from the sterilized water, large liquifiers were quite numerous. On the raw water these colonies were also present, but since a higher dilution was used, they were not so numerous,

Plates of the treated water have at times contained thousands of small colonies. These have made their appear- ance periodically. They are described later by chart.

A few of the monthly analyses and the average re-

duction in bacteria are shown in the following table:

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DATE 1913 Jan. Feb. March 3 March 23 april 42 May 3 May 12 July A July 14 Aug. Sept. Oct. Dec. 3 Dec. 30 1914 Jan.

Feb.

bacteria content.

R AW Agar Gelatin 770 2600 740 2200 70 5500 680 82000 530 6500 340 2600 1700 3800 233 50000 1200 1200 1500 6900 1250 3000 310 7600 1600 5100 86 430 320 3600 130 640

counting difficult.

This was often the case before the plates

STERILIZED Ager Gelatin Agar 10 85 6 47 60_ 12 20 7000 5 162 1240 6 90 155 0 43 760 6 35 36 3 800 7400 9700 33 65 34 250 220 97 114 300 210 76 260 45 325 550 70 36 35 a 43 52 55 110 2

had been incubated fifteen hours.

GENERAL EFFLUENT

Gelatin

116 340

These analyses show the effect of treatment on the

The presence of large liquefiers often made

After isolating them from

several different samples, they were found to be Bacillus

flourescens liquefacieus.

This organism is present in most

waters, both those on the surface and those from the ground.

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-27-

It was noticed here as in other cases that where liquefiers were present on gelation that the agar colonies would spread making accurate counting difficult.

Just how these bacteria get into the treated water is difficult to say. Apparently, all are not killed by hypo- chlorite. They may develop in some way in a part of the plant after the water has been filtered.

The following table gives results which were obtained by treating the strain with hypochlorite. The counts were

made from agar incubated at 37°C.

Available Chlorine Untreated Treated (pts.per mil)

me) 121 118

ol 170 170

08 98 105

24 120 116

26 230 225

.8 60 75 \ 1.0 200 190 1.2 175 100 1.4 150 130

The results show that this strain is not affected by calcium hypochlorite. Filtration does not entirely re- move them for they are found in the sterilized water and in the general effluent. They have no sanitary significance

and so can be looked on merely as peculiarities of this water.

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On the same water there developes at times thou- sands of minute colonies. These grow slowly at 20°C on gelatin. When these appeared on the gelatin plates colonies were taken off and put on agar slants. After the culture was purified, the characteristics were determined on the Standard media. The attached chart shows them. This organism is not a gas form and so is not a member of the colon group. According to Migula'’s classification it has

the characteristics of Micrococcus candicans.

PN EAE TT PTY RE EE STEN TPS RC PS NT

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SoureePekiirn..Well Wate. r.pdate OLAISOISNO Moye as a kt Name Bacillus Co./@./7......Group No. ()

NOTE—Underscore required terms.

DETAILED FEATURES. Observe notes

and glossary of terms on opposite side of card.

q Agar Hanging-Block

I. 1.

Hanging-Bleck

Il. a.

MORPHOLOGY (2) Vegetative Cells, Medium used.. A 3 g ALE...

temp. . Pay Ag vee, age. BF AP Sings

Form, short rods, long rods, short chains, long chains, filaments, commas, short spirale, long spirals, clostridium, cuneate, clavate,

curved. Limits of size. /A4.X Bu Size of Majority...."/.... Ends, rounded, truncate, concave. Orientation (grouping).........++ Chains (No. of elements)....... ° fa. lala nie tai a rientation o ains, parallel, irregular. medium used..

piace ala =... a

ag ays Form, elliptical, short rods, spindled, clavate,

drumsticks. Limits of Size... Size of Majority........

Orientation (grouping)........ a

Chains (No. of elements)......

Orientation of Chains, para

irregular.

Location of Endospores, central, polar.

Endospores,

Form, round, elliptical, elongated.

Limits of Size........ aes

Size of Majority.........

Wall, thick, thin.

Sporangium wall, adherent, not adherent.

Germination, equatorial, oblique, polar, by stretching. :

Flagella No,......Attachment polar, bipolar, per-

itrichiate. How Stained............

Capsules, present on............... Zoogloea, Pseudozoogloea.

Involution Iorms, on......... in...days at...9°C, Staining Reactions.

1:10 watery fuchsin, gentian violet, carbol fuchsin, Loeffler’s alkaline methylene blue.

Special Stains

CPM i aeucieigswiae GY CORED .. 2. ccceccccers .

Mauaetveteerttes---s-ees Acid fast ..... B piveies

NGUREED a atal ste sivrait aiele s

CULTURAL FEATURES (3)

Agar Stroke,

Growth, invisible, scanty, ae abundant.

Form of growth, —— echinulate, beaded, spreading, plumoseé, arborescent, rhizoid.

Elevation of growth, /lat, effuse, raised, conver.

Lustre, glistening, dull,

Topography, smooth,

Optical Characters, escent, iridescent.

Chromogenesis (8)

Odor, absent, decided, resembling...... seeees

Consistency, slimy, butyrous, viscid, membranous, coriaceous brittle,

Medium grayed, browned, reddened, blued, greened.

Potato. scanty, moderate, abundant, transient,

Growth, persistent. ; Form of growth, filiform, echinulate, beaded, plumose, arborescent, rhizoid. Elevation of growth, flat, effuse, raised, conver. Lustre, glistening, dull, cretaceous. Topography, smooth, contoured, rugose, verrucose,

Chromogenesis (8)......... +» Pigment in water insoluble, soluble; other solvents....... dle c' staete

Odor, absent, decided, resembling.............005 Consistency, slimy, butyrous, viscid, membranous, eereceous, mre. greneaneties

edium grayed, Hew reddened, blued, greened.

Loeffler’s Blood Serum.

Stroke invisible, scanty, moderate, abundant. Form of growth, filiform, echinulate, beaded, spreading, plumose, arborescent, rhizoid,

Elevation of growth, fiat, effuse, raised, conven.

Lustre, glistening, dull, cretaceous.

Topography, smooth, contoured, rugose, verrucose.

Chromogenesis (8)......... Medium grayed, browned, reddened, blued, greened.

Liquefaction begins in..... d, complete in...... d. Agar Stab. Growth uniform,

Sporangia,

ween eeee

Agar

bipolar,

rel, rugose,

opaque,

verrucose. translucent, opal-

best at bottom; sur-

face growth ae ddundant ; restricted, wide- spread, Line of puncture.

— wu. beaded, papillate, vil- lous, plumose, arborescent: liquefaction.

5.

Gelatin Stab. ; Growth yniform, , best at bottom.

best-at top Line of puncture, = beaded, papillate, vil lous, plumose, arborescent.

Liquefaction, crateriform, napiform, infundibuli- form, saccate, stratiform; begins in......... d, eomplete in......... d.

Medium fluorescent, browned......+.++++

Nutrient Broth.

Surface growth, ring, pellicle, flocculent, mem-

Gling abou mile te. at t t ouding slight, ate, strong; ransient, persistent; none; flur urbid.

Odor, absent, decided, resembling..........esees06

Sediment, compact, flocculent, granular, flaky,

Me on agitation, abundant, scant, ilk. ae a Clearing without coagulation. Coagulation _prompt, delayed, absent.

Extrusion of whey begins in..... coien OES Coagulum slowly peptonized, rapidly peptonized.

Peptonization begins on..... d, complete on..... d

Reaction, 1d..., 24.4.0 4d...., 10d...., 20d.... Consistency, slimy, viscid, unchanged,

Medium browned, reddened, blued, greened.

Lab ferment, present, absent.

Litmus Milk. r

Acid, alkaline, acid then alkaline, no change.

Prot reduction, no reduction, partial slow re- UCTLON,

9. ees se pane 4 rowth, slow, 1 3 k Form, Rare _ irregular, ameboid, mycelioid, filamentous, rhizoid. : Elevation, at. e/fuse, raised, conver, pulvinate, crateriform (liquefying). Edge, entire, undulate, lobate, erose, lacerate, fimbriate, filamentous, floccose, curled. 10 Tidnemcnen: cup, saucer, spreading.

. Agar Colonies. o Growth, slow, rapjd, temperature. . 9. 7.0.&. ; Form, punctiform, round, | irregular, ameboid,

mycelioid, filamentous, rhizoid. : 5 Surface smooth, rough, concentrically ringed, radi- ate, striate. , é Elevation, flat,- effuse, raised, conver, pulvinate, umbonate. Edge, entire, undulate, lobate, erose, lacerate, jimbriate, floccose, curled. Internal structure, amorphous, finely-, coarsely- granular, grumose, filamentous, floccose, curled. 11. Starch Jelly. , Growth, scanty, copious. Diastasic action, absent, feeble, profound. Medium stained.........-seeee 12, Silicate Jelly (Fermi’s Solution). Growth copious, scanty, absent. Medium stained........ alas daaings . 13. Cohn’s Solution. Growth, copious, scanty, absent. Medium fluorescent, non-fluorescent. 14, Uschinsky’s Solution. Growth copious, scanty, absent. Iluid viscid, not viscid. 15. Sodium Me in et ae Per cent inhibiting growth.......++sse-e0+ 16. Growth in Bouillon over Chloroform, wnre- strained, feeble, absent. : 17. Nitrogen. Obtained from peptone, asparagin, glycocoll, urea, ammonia salts, nitrogen. 18. Best media for long-continued growth.....-..+--- 19. Quick tests for differential purposeS....---+...++- III. PHYSICAL AND BIOCHEMICAL FEATURES. Fees Co) lll | le

Fermentation-Tubes con- | 2) Blo! oe |

taining peptone-water or | £/.4| 6/6 o's

Sugar-free bouillon and }/%/5 6 =| 5)

vo} | os! Sls ij ; Alan s|\OS| | Sl Gas production, in per cent. a lee ee ae (cor) | COs jee s Growth inclosed arm (Nag Me | ||| { id producedid. oo ails | Amount of acid p od Slofs] gt || “ oo “ od. | ay io awe ae “ van | | Et

2. Ammonia production, feeble, moderate, strong, absent, masked by acids. 3. Nitrate in nitrate broth, Reduced, not reduced. Presence of nitrites........ ABNOR... cee cece Se M MMEVATERS «cic ess free nitrogen......... 4. Indol production, feeble, moderate, strong. 5. Toleration of Acids: Great, medium, slight. At TERTEDS. wie esses. 6. Toleration of NaOH: Great, medium, slight. 7. Optimum reaction for growth in _ bouillon, stated in terms of Fuller's scale...........+.++ 8. Vitality on culture media: brief, moderate, long. 9. Temperature relations:

Thermal death-point (10 minutes exposure in nutrient broth when this is adapted to growth of GFZADISMA) «vg wne cs C.

Optimum temperature for growth........ C.: or best growth at 15° ©, 20° C, 25° C, 30° C, 87° C, 40° C, 50° C, 60° C.

Maximum temperature for growth........ Cc.

Minimum temperature for growth......... Cc.

10. Killed readily by drying: resistant to drying. 11. Per cent killed by freezing (salt and crushed ice or liquid air) ...i.s..eeees ae 12. Sunlight: Exposure on ice in thinly sown agar plates: one-half plate covered (time 15 min- utes), sensitive, not sensitive. Per cent killed). ..........+0% P 18, Acids produced ............ de 14. Alkalies produced ............. 15. Alcohols 2c .2c cc ccc ccc cose cue 16. Ferments; pepsin, trypsin, diastase, invertase, pectase, cytase, tyrosinase, owidase, peroridase, lipase, catalase, glucase, galactase, lab, etc..... 17. Crystals formed: ........eceeeeeeereeeeeceeceeeeees 18. Effect of germicides: B\2s5 o | = |SE ee a i/o 2) 2/56 Substance Method used. al e| 2 oa ££) S | 3 a) sh &\/E\|S\Ea i = | | aire ; sae IV. PATHOGENICITY.

1. Pathogenic to Animals

AO OP ~

Insects, crustaceans, fishes, reptiles, birds, mice,rats, guinea pigs, rabbits, dogs, cats, sheep, goats, cattle, horses, MONKEYS, MAM. ..ece.eceeeeeeees

. Pathogenic to Plants :

. Toxins, soluble, endotoxins, . Non-toxin forming.

. Immunity bactericidal.

. Immunity non-bactcricidal. . Loss of virulence on

culture media: prompt, sradual, not observed in--+-++-++++++e+++5- months.

BRIEF CHARACTERIZATION Mark + or O, and when two terms occur on a line erase the one which does not apply unless both apply.

‘SAUNLVaAA TVOINSHOOId

3 | Diameter over 1 = S Chains, filaments a ok 1 | Endospores = | Capsules ae S Zoogloea, Pseudozoogloea 5 ere : Motile * | Motite_ = S Involution forms Gram’s Stain = Cloudy, turpid +. F Ring Rate = | Pellicle = Sediment | Es Shining i ool Pr Dull eat a| & | Wrinkled |—_ cq Tepe > = _ Chromogenic esse a |, Round lt =| @ | Proteus-like - | |_Rhizoid |= | & | Filamentous |— led —— = | a Curled hee a 98 | Surface-growth = | + ~ | o | Needle-growth -b @ | Moderate, absent = [-- g Abundant p eee = | Discolored a Starch destroyed = Grows at 37° C. =

Grows, in Cohn’s Sol.

Luminous

Grows in Uschinsky’s Sol.

| Gelatin |— 22 | Blood-serum i— od s ia Casein 5 it ‘ | Agar, mannan |— | Acid curd +# = | Rennetcurd | “ |

Casein peptozized

Indoi (5) Hydrogen sulphide

~ Ammonia ©) Nitrates reduced (5) Fluorescent

NOILNEILSIG

| Fresh water

Iron bacterium

| Animal pathogen, epizoon

| Plant pathogen, epiphyte |

~ Soil Milk

"Salt water

Sewage

Sulphur bacterium

DESCRIPTIVE CHART—-SOCIETY OF AMERICAN

Prepared by F. D. CHESTER

F

P. GORHAM

ERWIN F. SMITH

BACTERIOLOGISTS

Committee on Methods of Identification of Bacterial Species.

ENDORSED BY THE SOCIETY FOR GENERAL USE AT THE ANNUAL MEETING, DEC. 31, 1907.

GLOSSARY OF TERMS.

AGAR HANGING BLOCK, a small block of nutrient agar cut from a poured plate, and placed on a cover-glass, the surface next the glass having been first touched with a loop from a young fluid culture or with a dilution from the same. It is examined upside down, the same as a hanging drop.

AMEBOID, assuming various shapes like an ameba.

AMORPHOUS, without visible differentiation in structure.

ARBORESCENT, a branched, tree-like growth.

BEADED, in stab or stroke, disjointed or semi-confuent colonies along the line of inoculation,

BRIEF. a few days, a week.

BRITTLE, growth dry, friable under the platinum needle.

BULLATE, growth rising in convex prominences, like a blistered surface.

BUTYROUS, growth of a butter-like consistency.

CHAINS.

Short chains, composed of 2 to 8 elements. Long chains, composed of more than § elements,

CILIATE, having fine, hair-like extensions like cilia.

CLOUDY, said of fluid cultures which do not contain pseudozoogloeae,

COAGULATION, the separation of casein from whey in milk. This may take place quickly or slowly, and as the result either of the formation of an acid or of a lab ferment.

CONTOURED, an irregular, smoothly undulating surface, like that of a relief map. ;

CONVEX. surface the segment of a circle, but flattened.

COPROPHYL, dung bacteria.

CORIACEOUS, growth tough, leathery, not yielding to the platinum needle.

CRATERIFORM, round, depressed, due to the liquefaction of

the medium.

CRETACEOUS, growth opaque and white, chalky.

CURLED, composed of parallel] chains in wavy strands, as in anthrax colonies.

DIASTASIC ACTION, Same as DIASTATIC, conversion of starch into water-soluble substances by diastase.

ECHINULATE, in agar stroke a growth along line of inocula- tion. with toothed or pointed margins: in stab cultures growth beset with pointed outgrowths.

EFFUSE, growth thin, veily, unusually spreading,

ENTIRE, smooth, having a margin destitute of teeth or notches.

EROSE. border irregularly toothed.

FILAMENTOUS, growth composed of long, interwoven filaments.

FILIFORM, in stroke or stab cultures a uniform growth along line of inoculation.

FIMBRIATE, border fringed with slender processes, filaments.

FLOCCOSE, growth composed of short curved chains, oriented.

FLOCCULENT. said of fluids which contain pseudozcogloeac, i. e., small adherent masses of bacteria of various shapes and floating in the culture fluid.

FLUORESCENT, having one color by another by reflected light.

GRAM’S STAIN, a method of differential bleaching after gentian violet, methyl violet, etc. The + mark is to be given only when the bacteria are deep blue or remain blue after counterstaining with Bismark brown.

GRUMOSE., clotted.

INFUNDIBULIFORM, form of a funnel or inverted cone.

IRIDESCENT, like mother-of-pearl. The effect of very thin films.

LACERATE, having the margin cut into irregular segments as if torn.

LOBATE, border deeply undulate, producing lobes (see undulate.)

LONG. many weeks.

irregularly placed or

larger than

variously

transmitted light and

or months,

MAXIMUM TEMPERATURE, does not take place,

MEDIUM, several weeks.

MEMBRANOUS, growth thin, coherent, like a membrane.

MINIMUM TEMPERATURE, temperature below which growth does not take place. :

MYCELIOID. colonies having the radiately filamentous appear- ance of mold colonies,

NAPIFORM, liquefaction with the form of a turnip.

NITROGEN REQUIREMENTS, the necessary nitrcgenous focd. This is determined by adding to nitrogen-free media the nitrogen compound to be tested.

OPALESCENT, resembling the color of an opal.

OPTIMUM TEMPERATURE, temperature at which growth is most rapid,

PELLICLE, in fluid bacterial growth either forming a continuous or an interrupted sheet over the fluid.

PEPTONIZED, said of curds dissolved by trypsin.

PERSISTENT, many weeks, or months,

PLUMOSE, a fleecy or feathery growth.

PSEUDOZOOGLOEAE, clumps of bacteria, not dissolving readily in water, arising from imperfect separation, or more or less fusion of the components, but not having the degree of compactness and gelatinization seen in zoogloeae.

PULVINATE, in the form of a cushion, decidedly convex,

PUNCTIFORM, very minute colonies, at the limit of natural vision.

RAISED, growth thick, with abrupt or terraced edges.

RHIZOID, growth of an irregular branched or root-like character, as in B. mycoides.

RING. Same as RIM, growth at the upper margin of a liquid culture, adhering more or less closely to the glass.

REPAND. wrinkled.

RAPID, Developing in 24 to 48 hours.

SACCATE, liquefaction the shape of an elongated sack, tubular, cylindrical.

SCUM, floating islands of bacteria, bacterial membrane,

SLOW. requiring 5 or 6 days or more for development.

SHORT, applied to time, a few days, a week.

temperature above which growth

an interrupted pellicle or

SPORANGIA, cells containing endospores,

SPREADING, growth extending much beyond the line of inoculation, i. e., several millimeters or more.

STRATIFORM, liquefying to the walls of the tube at the top

and then proceeding downwards horizontally.

THERMAL DEATH-POINT, the degree of heat required to kill young fluid cultures of an organism exposed for 10 minutes (in thin-walled test tubes of a diameter not exceeding 20 mm.) in the thermal water-bath. The water must be kept agitated so that the temperature shall be uniform during the exposure.

TRANSIENT, a few days,

TURBID. cloudy with flocculence.

UMBONATE, having a button-like. raised center.

UNDULATE, border wavy. with shallow sinuses.

VERRUCOSE, growth wart-like, with wart-like prominences.

VERMIFORM-CONTOURED, growth like a mass of worms, or intestinal coils.

VILLOUS, growth beset with hair-like extensions,

VISCID. growth follows the needle when touched and withdrawn, sediment on shaking rises as a coherent swirl.

ZOOGLOEAE, firm gelatinous masses of bacteria, one of the most typical examples of which is the Streptococcus mesenter- ioides of sugar vats (Leuconostoc mesenterioides), the bac- terial chains being surrounded by an enormously thickened firm covering, inside of which there may be one or many groups of the bacteria.

flocculent particles; cloudy plus

NOTES.

(1) For decimal system of group numbers see Table 1. This will be found useful as a quick method of showing close rela- tionships inside the genus, but is not a sufficient characterization of any organism.

(2) The morphological characters shall be determined and described from growths obtained upon at least one solid medium (nutrient agar) and in at least one liquid medium (nutrient broth). Growths at 37° C shall be in general not older than 24 to 48 hours, and growths at 20° C not older than 48 to 72 hours. To secure uniformity in cultures, in all cases preliminary cultiva- tion shall be practiced as described in the revised Report of the Committee on Standard Methods of the Laboratory Section of the American Public Health Association, 1905.

(3) The observation of cultural and bio-chemical features shall cover a period of at least 15 days and frequently longer, and shall be made according to the revised Standard Methods above referred to. All media shall be made according to the same Standard Methods.

(4) Gelatin stab cultures shall be held for 6 weeks to deter- mine liquefaction. -

(5) Ammonia and indol tests shall be made at end of 10th day, nitrite tests at end of 5th day.

(6) Titrate with N NaOH, using phenolphthalein as an

20 indicator: The

make titrations at same times from blank. difference gives the amount of. acid produced.

The titration should be done after boiling to drive off any Co2 present in the culture, i

(7) Generic nomenclature shall begin with the year 1872 (Cohn’s first important paper).

Species nomenclature shall begin with the year 1880

(Koch’s discovery of the poured plate method for the separation of organisms).

(8) Chromogensis shall be recorded in standard color terms.

TABLE I.

A NUMERICAL SYSTEM OF RECORDING THE SALIENT CHARACTERS OF AN ORGANISM. (GROUP NUMBER.)

100. Endospores produced 200. Endospores not produced ; 10. Aerobie (Strict) ; 20. Facultative anaerobic 30. Anaerobic (Strict)

as Gelatin liquefied

2. Gelatin not liquefied

0.1 Acid and gas from dextrose

0.2 Acid withext gas from dextrose

0.3 No acid from dextrose

0.4 No growth with dextrose

.O1 Acid and gas from lactose

.02 Acid without gas from lactose

-03 No acid from lactose

-04 No growth with lactose

001 Acid and gas from saccharose

002 Acid without gas from saccharose

.003 No acid from saccharose

-004 No growth with saccharose :

0001 Nitrates reduced with evolution of gas

-0002 Nitrates not reduced

-0003 Nitrates reduced without gas formation

-00001 Fluorescent

00002 Violet chromogens

00003 Blue a

00004 Green 43

-00005 Yellow “s

-00006 Orange a4

00007 Red Ud

00008 Brown ae

-00009 Pink ye

.00000 Non-chromogenic

-000001 Diastasie action on potato starch, strong

.000002 Diastasic action on potato starch, feeble

000003 Diastasic action on potato starch, absent

0000001 Acid and gas from glycerine

eeiecs Acid without gas from glycerine

d 10038

No acid from glycerine ij -0000004 No growth with glycerine :

The genus according to the system of Migula is given its proper symbol which precedes the number thus: (7)

BACILLUS COLI (Esch.) Mig. becomes B. 222,111102 BACILLUS ALCALIGENES Petr. co B. 212.3331! PSEUDOMONAS CAMPESTRIS (Pam.) Sm. ** Ps. 211.333151

BACTERIUM SUICIDA Mig. Jd Bact, 222,232208

“4

ee a a ee ee

Sano A I TE IED BIT oS SE ET AE

DETAILED FEATURES.

NOTE—Underscore required terms. Observe notes

and glossary of terms on opposite side of card.

I. MORPHOLOGY (2) : 1. Vegetative Cells, Medium used. A GF AS.... MECINDe twee tess. >, ALC. ...00s toe GSyS Form, round, short rods, long rods, short chains, long chains, filaments, long _ spirals,

_ curved. Limits of Stye.:.f, Aus.

Size of Majority.4.......

Ends, rounded, truncate, concave. : Orientation (grouping)........ rei Chains (No. of elements)....... ‘i Short chains, long chains Orientation of Chains, irregular.

medium used..........

commas,

8, short spirals, clostridium,

cuneate, clavate,

Agar Hanging-Block parallel, 2. Sporangia,

(Pe)

ag . days Form, elliptical, short rods, spindled, clavate,

drumsticks.

Limits of Size.. Size of Majority...... ee Orientation (grouping)......

Chains (No. of elements)......

Orientation of Chains, parallel,

irregular,

Agar Hanging-Bleck

Location of Endospores, central, polar. 3. ‘Endospores,

Form, round, elliptical, elongated.

‘Limits of Size......... Ao

Size of Majority.........

Wall, thick, thin.

Sporangium wall, adherent, not adherent.

Germination, equatorial, oblique, polar, bipolar, by stretching. 4. Flagella No.......Attachment polar, bipolar, per- itrichiate. How Stained............ 5. Capsules, present on............... 6. Zoogloea, Pseudozoogloea. 7. Involution Forms, on......... in...days at...°C, 8. Staining Reactions. 1:10 watery fuchsin, gentian violet, carbol fuchsin, Loeffler’s alkaline methylene blue. Special Stains Pe aie Penivigiwes SGHLY. COPED . oss nacuaece cous iiiestaeieieviae cece sweees ACIG faSt......00.0secees IWGIBSED )eeiscctirisvisclensais Il, CULTURAL FEATURES (3)

1. Agar Stroke. Growth, invisible, scamty moderate, abundant. Form of growth, i , echinulate, beaded, spreading, plumose, arborescent, rhizoid. Elevation of growth, flat, effuse, raised, conver. oe gihatentng. dull, cretacecous. opography, —S Optical Characters, escent, iridescent. Chromogenesis (8)

Odor, absent, decided, resembling....... seeats Consistency, slimy, butyrous, viscid, membranous, coriaceous brittle. Medium grayed, browned, reddened, blued, greened. 2. Potato,

contoured, rugose, verrucose.

opaque, translucent, opal-

Growth, .scanty, moderate, abundant, transient, persistent. : Form of growth, filiform, echinulate, beaded,

spreading, plumose, arborescent, rhizoid. Elevation of growth, effuse, raised, conve. Lustre, glistening, dull cretaceous. Topography, smooth, contoured, rugose, verrucose,

Chromogenesis (8) Bf elle Pigment in water

me

BRIEF CHARACTERIZATION Mark + or O, and when two terms

occur on a line erase the one which

does not apply unless both apply.

OW

(@ ‘ADOTOHdY

Diameter over 1/4

—_

Chains, filaments

Endospores

Capsules

Zoogloea, Pseudozoogloea

Motile

Involution forms

Gram’s Stain

(8) ‘SAYNLVaA TVANLITNO

Cloudy, turpid Ring = Pellicle Sediment

qjo1g

Shining

Dull Wrinkled Chromogenic

Ivsy

Round

Proteus-like

Rhizoid

Filamentous

381d “19D

: Curled

Surface-growth

Needle-growth

Moderate, absent

Abundant

Discolored Starch destroyed

Grows at 37° C.

Grows, in Cohn’s Sol.

Grows in Uschinsky’s Sol.

‘SAUNLVAA TVOINEHOOITA

fie Wager... >: ia Date of Isolation................. 5. neiee Name Micrecaceus.C aaG'CGriaup No. (1) 5. Gelatin Stab. 2. Ammonia roduction, feeble, moderate, strong, Growth uniform, best at top, best at bottom. ehrent ih edb aie 4 “ Line of puncture, jlliform, beaded, papillate, vil 4 a hi 1 a atk lous, plumose, arborescent. 3. Nitrate in nitrate broth, Liquefaction, crateriform, napiform, infundibuli- Reduced, not reduced. form, saccate, stratiform; begins in.........d, Presence of nitrites........ @MIMONIA. .. 20 cece aie complete in......... d. <3 Se TMGEALESS oes «css free nitrogen......... a eee ae acer browned......+.. eee 4. Indol production, feeble, moderate, strong. . Nutrien roth, 2 are 4 fs Surface growth, ring, pellicle, flocculent, mem- 5. Toleration of Acids: Great, medium, slight. branous, none. } Acids tested............... erent nent ee transient, 6. Toleration of NaOH: Great, medium, slight. Odor, qhsent, decided, resembling........ 6.00.05 7. Optimum reaction for growth in bouillon, Sediment, CouLpat he feetedea) granenar, flaky, stated in terms of Fuller’s scale............--. 7. Be aa on agitation, abundant, scant. 8. Vitality on culture media: brief, moderate, long. Clearing without coagulation. 9. Temperature relations: Coagulation prompt, delayed, absent. Thermal death-point (10 minutes exposure in Extrusion of whey begins in..... rage SOON Ge se nutrient broth when this is adapted to growth of Coagulum slowly peptonized, rapidly peptonized. organism).......... Cc. Peptonization begins on..... d, complete on....-. d Optimum temperature for growth........ C.: or Reaction, dvs.) 2der-, 4d-..., 10d...., 20d.... best growth at 15° C, 20° C€, 25° ©, 30° C, Consistency, slimy, viscid, unchanged. 37° C, 40° C, 50° C, 60° C. Medium browned, ee eee greened. Maximum temperature for growth........ Con 8. ye he present, absent. Minimum temperature for growth........- ce Acid, alkaline, acid then alkaline, no change. 10. Killed readily by drying: resistant to drying. Promel TSaaetet: ng Peduoninn, (partial ate. ee 11. Per cent killed by freezing (salt and crushed ice 9. Gelatin Colonies. or Jiqnid air)..-.........8 . Growth, slow, rapid, ‘ 12. Sunlight: Exposure on ice in thinly sown agar Form, unctiform, irregular, ameboid, sara . ran Tceleld, filamentous, rhizoid. ; plates: one-half plate covered (time 15 min Elevation, flat, effuse, raised, convex, pulvinate, utes), sensitive, not sensitive. crateriform (liquefying). RUE tee Tots dices ae Edge, utire, undulate, lobate, erose, lacerate, Per Sob! killed fimbriate, filamentous, floccose, curled. 13, Acids produced ............0++ 10 TAgmctactions: cup, saucer, spreading. 14. Alkalies produced ............+ PA Sa ace d5o Ateohols®. occsccsss----ss+.. am Growth, slow, rapid, temperature......+.-+-- : . epens : ; : Form, Bh bac round. irregular, ameboid, 16. Ferments; pepsin, trypsin, diastase, invertase, mycelioid, filamentous, rhizoid. od di pectase, cytase, tyrosinase, oxidase, perovidase, ar RONG Nas CIRC EOS VOY: KINIEE. TAEY> lipase, catalase, glucase, galactase, lab, etc..... Elevation, flat, effuse, raised, conver, pulvinate, ne aeecaceeeereeerebeeeeeeeesececeeeres umbonate. 17. Crystals formed: ........--Becsceccescecceceneccees Edge, entire, undulate, lobate, erose, lacerate, " ; jimbriate, floccose, curled. 18. Effect of germicides: Internal structure, amorphous, finely-, coarsely- granular, grumose, filamentous, floccose, curled. tl 11. Starch Jelly. 4 Biles Growth, scanty, copious. " = 35 Diastasic action, absent, feeble, profound. E/ sige Medium stained.......++--+++ eS 3h 12. Silicate Jelly (Fermi’s Solution). Substance Method used. al - oe Growth copious, scanty, absent. 2 3 eee Medium stained........seeees = ale a5 13. Cohn’s Solution. =) o/s \ES Growth, copious, scanty, absent. Slal My lan Medium fluorescent, non-fluorescent. : 14. Uschinsky’s Solution. Growth copious, scanty, absent. 5 ‘ 1 Fluid viscid, not viscid. 15. Sodium Chloride in Bouillon. - | Per cent inhibiting growth.........-sssees 16. Growth in Bouillon’ over Chloroform, wnre- eee gal (ae strained, feeble, absent. : 17. Nitrogen. Obtained from peptone, asparagin, ft 3 ee et glycocoll, urea, ammonia salts, nitrogen. | 18. Best media for long-continued growth. ......-+++: = = 7 a 19. Quick tests for differential purposes...-----+--+-- ee See —— USS Ee AEE agese cog eee pete | . = ase haltisle Seat iy Aeriers se eee, III. PHYSICAL AND BIOCH IV. PATHOGENICITY.

insoluble, soluble; other solvents..... Ay

Odor, absent, decided, resembling..........+..++.

Consistency, slimy, butyrous, viscid, membranous, coriaceous, brittle.

Medium grayed, browned, reddened, blued, greened.

3. Loeffler’s Blood Serum.

Stroke invisible, scanty, moderate, abundant. Form of growth, filiform, echinulate, beaded, spreading, plumose, arborescent, rhizoid.

Elevation of growth, flat, effuse, raised, convez,

Lustre, glistening, dull, cretaceous.

Topography, smooth, contoured, rugose, verrucose.

“Chromogenesis (8)......... Medium grayed, browned, reddened, blued, greened. Liquefaction begins in..... d, complete in...... d.

4. Agar Stab.

Growth uniform, best at top, best at bottom; sur- face alia scanty, abundant; restricted, wide- spread.

Line of puncture. filiform, beaded, papillate, vil- lous, plumose, arborescent: liquefaction.

Gas production, in per cent. |

o an . ;o|O rs] Fermentation-Tubes con- | 4) §| 0) 5). taining peptone-water or £/.4) 6) 9/ 9/6 Sugar-free bouillon and | % = Sic SS Sl asim Alal A =|Ol=

Growth in closed arm

Amount of acid producedi d. fol

( cos) No Gals |_|

“ ok “

2d. |

1, Pathogenic to Animals

“1D Ol em OO

ET CTE TY REY ATI RET LIS LETT TET ERTIES ST EE I TIRE LI SEPT EE

Insects, crustaceans, fishes, reptiles, birds, mice,rats, guinea pigs, rabbits, dogs, cats, sheep, goats, cattle, horses, MONREYS, MAN ..++++...e--eee eee

. Pathogenic to Plants :

. Toxins, soluble, endotoxins,

. Non-toxin forming.

. Immunity bactericidal.

. Immunity non-bactericidal.

. Loss of virulence on culture

media: prompt, Sradual, not observed im---++-++++++++++- months.

NOILNALALSIG

Gelatin Ole

Blood-serum

Casein

uo} -ovjonbryT

| Agar, mannan

Acid curd

TON

Rennet curd

Hydrogen sulphide

Ammonia 6)

Nitrates reduced (5)

Fluorescent

Luminous

| Animal pathogen, epizoon

Plant pathogen, epiphyte |

_ Milk

Soil

Fresh water

~ Salt water

Sewage

| Tron bacterium

/ Sulphur bacterium

DESCRIPTIVE CHART—SOCIETY OF AMERICAN BACTERIOLOGISTS

Prepared by F. D. CHESTER

F P. GORHAM ERWIN F. SMITH

ENDORSED BY THE SOCIETY FOR GENERAL USE AT THE ANNUAL MEETING, DEC. 31, 1907.

Committee on Methods of Identification of Bacterial Species,

GLOSSARY OF TERMS.

AGAR HANGING BLOCK, a small block of nutrient agar cut from a poured plate, and placed on a cover-glass, the surface next the glass having been first touched with a loop from a young fluid culture or with a dilution from the same. It is examined upside down, the same as a hanging drop.

AMEBOID, assuming various shapes like an ameba.

AMORPHOUS, without visible differentiation in structure.

ARBORESCENT, a branched, tree-like growth.

BEADED, in stab or stroke, disjointed or semi-confuent colonies along the line of inoculation,

BRIEF. a few days, a week.

BRITTLE, growth dry, friable under the platinum needle.

BULLATE, growth rising in convex prominences, like a blistered surface.

BUTYROUS, growth of a butter-like consistency.

CHAINS, -

Short chains, composed of 2 to 8 elements. Long chains, composed of more than 8 elements.

CILIATE, having fine, hair-like extensions like cilia.

CLOUDY, said of fluid cultures which do not contain pseudozoogloeae.

COAGULATION, the separation of casein from whey in milk. This may take place quickly or slowly, and as the result either of the formation of an acid or of a lab ferment.

CONTOURED, an irregular, smoothly undulating surface. like that of a relief map.

CONVEX. surface the segment of a circle. but flattened,

COPROPHYL, dung bacteria.

CORIACEOUS, growth tough, leathery, not platinum needle.

CRATERIFORM, round, depressed, due to the liquefaction of the medium.

CRETACEOUS, growth opaque and white, chalky.

CURLED, composed of parallel] chains in wavy strands. as in anthrax colonies.

DIASTASIC ACTION, Same as DIASTATIC, conversion of starch into water-soluble substances by diastase.

ECHINULATE, in agar stroke a growth along line of inocula- tion. with toothed or pointed margins; in stab cultures growth beset with pointed outgrowths.

EFFUSE, growth thin, veily, unusually spreading.

ENTIRE. smooth, having a margin destitute of teeth or notches.

EROSE. border irregularly toothed.

FILAMENTOUS, growth composed of long, irregularly placed or interwoven filaments.

FILIFORM, in stroke or stab cultures a uniform growth along line of inoculation.

FIMBRIATE, border fringed with slender processes, larger than filaments.

FLOCCOSE, growth composed of short curved chains, variously oriented.

FLOCCULENT, said of fluids which contain pseudozcogloeac, i. e., small adherent masses of bacteria of various shapes and floating in the culture fluid,

FLUORESCENT. having one color by transmitted light and another by reflected light.

GRAM’S STAIN, a method of differential bleaching after gentian violet, methyl violet, etc. The + mark is to be given only when the bacteria are deep blue or remain blue after counterstaining with Bismark brown.

GRUMOSE, clotted.

INFUNDIBULIFORM, form of a funnel or inverted cone.

: IRIDESCENT, like mother-of-pearl. The effect of very thin films.

yielding to the

LACERATE, having the margin cut into irregular segments as if torn.

TOBATE, border deeply undulate, producing lobes (see undulate.) LONG, many weeks, or months.

MAXIMUM TEMPERATURE, temperature above which growth does not take place,

MEDIUM, several weeks.

MEMBRANOUS, growth thin, coherent, like a membrane.

MINIMUM TEMPERATURE, temperature below which growth does not take place.

MYCELIOID. colonies having the radiately filamentous appear- ance of mold colonies.

NAPIFORM, liquefaction with the form of a turnip.

NITROGEN REQUIREMENTS, the necessary nitregenous focd. This is determined by adding to nitrogen-free media the nitrogen compound to be tested.

OPALESCENT, resembling the color of an opal.

OPTIMUM TEMPERATURE, temperature at which growth is most rapid.

PELLICLE, in fluid bacterial growth either forming a continuous or an interrupted sheet over the fluid.

PEPTONIZED, said of curds dissolved by trypsin,

PERSISTENT, many weeks, or months,

PLUMOSE, a fleecy or feathery growth.

PSEUDOZOOGLOEAE, clumps of bacteria, not dissolving readily in water, arising from imperfect separation, or more or less fusion of the components, but not having the degree of compactness and gelatinization seen in zoogloeae.

PULVINATE, in the form of a cushion, decidedly convex,

PUNCTIFORM, very minute colonies, at the limit of natural vision,

RAISED, growth thick, with abrupt or terraced edges.

RHIZOID, growth of an irregular branched or root-like character, as in B. mycoides.

RING. Same as RIM, growth at the upper margin of a liquid eulture, adhering more or less closely to the glass,

REPAND. wrinkled.

RAPID, Developing in 24 to 48 hours.

SACCATE, liquefaction the shape of an elongated sack, tubular, cylindrical.

SCUM, floating islands of bacteria, an interrupted pellicle or bacterial membrane,

SLOW, requiring 5 or 6 days or more for development.

SHORT, applied to time. a few days. a week.

SPORANGIA, cells containing endospores.

SPREADING, growth extending much beyond the line of inoculation, i. e., several millimeters or more.

STRATIFORM, liquefying to the walls of the tube at the top and then proceeding downwards horizontally.

THERMAL DEATH-POINT, the degree of heat required to kill young fluid cultures of an organism exposed for 10 minutes (in thin-walled test tubes of a diameter not exceeding 20 mm.) in the thermal water-bath. The water must be kept agitated so that the temperature shall be uniform during the exposure.

TRANSIENT, a few days,

TURBID. cloudy’ with flocculence.

UMBONATE, having a button-like, raised center.

UNDULATE. border wayy. with shallow sinuses.

VERRUCOSE, growth wart-like, with wart-like prominences.

VERMIFORM-CONTOURED, growth like a mass of worms, or intestinal coils.

VILLOUS. growth beset with hair-like extensions,

VISCID. growth follows the needle when touched and withdrawn, sediment on shaking rises as a coherent swirl.

ZOOGLOEAE, firm gelatinous masses of bacteria, one of the most typical examples of which is the Streptococcus mesenter- ioides of sugar vats (Leuconostoc mesenterioides), the bac- terial chains being surrounded by an enormously thickened firm covering, inside of which there may be one or many groups of the bacteria.

flocculent particles; cloudy plus

NOTES.

(1) For decimal system of group numbers see Table 1. This will be found useful as a quick method of showing close rela- tionships inside the genus, but is not a sufficient characterization of any organism,

(2) The morphological characters shall be determined and described from growths obtained upon at least one solid medium (nutrient agar) and in at least one liquid medium (nutrient broth). Growths at 37° C shall be in general not older than 24 to 48 hours, and growths at 20° C not older than 48 to 72 hours. To secure uniformity in cultures. in all cases preliminary cultiva- tion shall be practiced as described in the revised Report of the Committee on Standard Methods of the Laboratory Section of the American Public Health Association, 1905.

(3) The observation of cultural and bio-chemical features shall cover a period of at least 15 days and frequently longer, and shall be made according to the revised Standard Methods above referred to. All media shall be made according to the same Standard Methods.

(4) Gelatin stab cultures shall be held for 6 weeks to deter- mine liquefaction,

(5) Ammonia and indol tests shall be made at end of 10th day, nitrite tests at end of 5th day.

(6) Titrate with N NaOH, using phenolphthalein as an

indicator: make titrations at same times from blank, The difference gives the amount of acid produced.

The titration should be done after boiling to drive off any Co2 present in the culture.

(7) Generic nomenclature shall begin with the year 1872 (Cohn’s first important paper). ;

Species nomenclature shall begin with the year 1880

(Koch's discovery of the poured plate method for the separation of organisms).

(8) Chromogensis shall be recorded in standard color terms.

TABLE I.

A NUMERICAL SYSTEM OF RECORDING THE SALIENT CHARACTERS OF AN ORGANISM. -(GROUP NUMBER.)

100. Endospores produced 200. Endospores not produced 10. Aerobie (Strict) 20. Facultative anaerobic 30. Anaerobic (Strict) 1. Gelatin liquefied 2; Gelatin not liquefied 0.1 Acid and gas from dextrose 0.2 Acid withovwt gas from dextrose 0.3 No acid from dextrose 0.4 No growth with dextrose 01 Acid and gas from lactose .02 Acid without gas from lactose .03 No acid from lactose . No growth with lactose 001 Acid and gas from saccharose -002 Acid without gas from saccharose 003 No acid from saccharose - 004 No growth with saccharose 0001 Nitrates reduced with evolution of gas -0002 Nitrates not reduced .0003 Nitrates reduced without gas formation 00001 Fluorescent -00002 Violet chromogens 00003 Blue oF -00004 Green 4 .00005 Yellow a 00006 Orange se 00007 Red a8 -00008 Brown ole -00009 Pink = -00000 Non-chromogenic -000001 Diastasic action on potato starch, stro! .000002 Diastasic action on potato starch, feeble 000003 Diastasic action on potato starch, absent 0000001 Acid and gas from glycerine .0000002 Acid without gas from glycerine .0000003 No acid from glycerine .0000004 No growth with glycerine

The genus according to the system of Migula 1

given its proper symbol which precedes the number thus: ( BACILLUS COLI (Esch.) Mig. becomes B, 222.11110 BACILLUS ALCALIGENES Petr. bY B. 212, PSEUDOMONAS CAMPESTRIS (Pam.) Sm.“ Ps. BACTERIUM SUICIDA Mig. a Bact. 222.

a

_ NOTE—Underscore required terms.

Source V1 ;

DETAILED FEATURES. Observe notes

and glossary of terms on opposite side of card.

1S 2

Agar Hanging-Block

2.

Agar Hanging-Bleck

3.

Bhs Zz,

4.

MORPHOLOGY (2) Vegetative Cells, Medium used.. A G AF...

Oey a. f Poon days

ge...

Form, round, short rods, long rods, long chains, filaments, commas, Shor long spirals, clostridium, cuneate,

curved. Eimite (Of Size... 1.2.00

Size of Majority.........

Ends, rounded, truncate, concave. Orientation (grouping)..........+ Chains (No. of elements)......

, long chains Orientation of Chains, pargllel irregular,

Sporangia, medium used..........temp....... Aes

Girsivts saieiuisin a

Form, elliptical, drumsticks.

Limits of Size......... Size of Majority........

Orientation (grouping)..........

Chains (No, of elements)......

Orientation of Chains, parallel,

irregular.

Location of Endospores, central, polar,

Endospores.

Form, round, elliptical, elongated,

DRT OL SIZE: Sec se.

Size of Majority.........

Wall, thick, thin.

Sporangium wall, adherent, not adherent.

Germination, equatorial, oblique, polar, bipolar, by stretching.

Flagella No.......Attachment polar, bipolar, per-

itrichiate. How Stained............

Capsules, present on............... Zoogloea, Pseudozoogloea.

Involution Forms, on......... in...days at...°C, Staining Reactions. 1:10 watery fuchsin, gentian violet, carbol fuchsin, Loeffler’s alkaline methylene blue. ecial Stains

DOM b ib ae selec <cspee SGIYCOPEN «0. rcecesee

PiAtciaesenees oa tioscces ACIA fast ....00.. veneers

EME MEL aici ts selatols.s\e/6 o)a'0.4 CULTURAL FEATURES (3)

Tawth ten idl t derat

rowth, invisible, scanty, moderate, Abundant,

Form of growth, filiform, echinulate, beaded, spreading, plumose, arborescent, rhizoid.

Elevation of growth, flat, effuse, raised, conver.

Lustre, glistening, dull, cretoccous

Topography,

Optical Characters,

spirals, clavate,

ys short rods, spindled, clavate,

contoured, rugose, verrucose. ha opaque, translucent, opal: , tridescen

cscent t Chromogenesis (8) Gree nesA. Odor, absent, decided, resembling....... preter Consistency, slimy, butyrous, viscid, membranous, coriaceous brittle. Medium grayed, browned, reddened, blued, greened.

Growth, scanty, persistent. 7

Form of growth, filiform, echinulate, beaded, spreading, plumose, arborescent, rhizoid,

Elevation of growth, flat, effuse, raised, convez.

Lustre, glistening, dull, cretaceous.

Topography, ee Beoa/p rugose, verrucose,

Chromogenesis (8) 9.1 Pigment in water insoluble, soluble; other solvents...... Ses “

Odor, absent, decided, resembling................

Consistency, slimy, butyrous, viscid, membranous, coriaceous, brittle.

Medium grayed, browned, reddened, blued, greened.

Loeffler’s Blood Serum.

Stroke invisible, scanty, moderate, abundant. Form of growth, filiform, echinulate, beaded, spreading, plumose, arborescent, rhizoid.

Elevation of growth, flat, effuse, raised, convez.

Lustre, glistening, dull, cretaccous.

Topography, smooth, contoured, rugose, verrucose.

Chromogenesis (8)......... Medium grayed, browned, reddened, blued, greened,

Liquefaction begins in..... d, complete in...... d. Agar Stab

Growth uniform, Dest ai-top. best at bottom; sur- face growth scanty, abundant; restricted, wide- _ spread,

Line of puncture. filiform, beaded, papillate, vil-

lous, plumose, arborescent: liquefaction.

moderate, abundant, transient,

SSS TT AP RR SES TT EN EM ES ROE ah RL AAT TE AE LS a EET,

Group No. (i)...

5. en Sieh. Nk ee SE AS 2. Ammonia productioh, feeble, moderate, ‘strong, BRIEF CHARACTERIZATION rowth uniform, best at top, best at bottom. ; zi Line of puncture, filiform, beaded, papillate, vil absent, masked by acids. Mark + or O, and when two terms lous, plumose, arborescent. 3. Nitrate in nitrate broth, sare ot a da ore the one which Liquefaction, crateriform, napiform, infundibuli- Reduced, not reduced. oes 20 apply unless both apply. form, sggcate, stratiform; begins in.........d, Presence of nitrites........ ATMBIB WIA y6.01cie.s) ctsre's)e 5 Diameter over 14 = complete in.......-. d. ve PSO SMTMALES oisisie is: « free nitrogen......... 5 | Chains, filaments aol Medium Dyoregcent, browned...... ar. 4. Indol production, feeble, moderate, strong. ee Endospores — 6. Nutrient : 5 4 5. Toleration of Acids: Great, medium, slight. | pel growth, ring, pellicle, flocculent, mem- hide eaiied iS} Canetles ae ranous, none. i fe Ee ea ee te le transient, 6. Toleration of NaOH: Great, medium, slight. 3 Zoogloea, Pseudozoogloea | — Odor, absent, decided, resembling........++++-+-++ 7. Optimum reaction for growth in_ bouillon, < Motile + Sediment, compact, flocculent, granular, flaky, stated in terms of Fuller’s scale..............- o a 7 ee on agitation, gbundant, scant. 8. Vitality on culture media: brief, moderate, long. © | _Involution forms ee Clearing without eee ah 9. Temperature relations: Gram’s Stain — latio t, delayed, absent. -poi fa ; = Coagulation promp y : Thermal death-point (10 minutes exposure in ; fnid + Extrusion of whey begins in..... ‘sole sGAyie nutrient broth when this is adapted to growth of ow - _|— Coagulum slowly peptonized, rapidly peptonized. organism).......... C. 4 Ring = Peptonization begins on..... d, complete on..... d Optimum temperature for growth........ C.: or =a Pellicle a s 5 5° C, 30° C - et Reaction, 1d..., 2d...., 4d...., 10d...., 20d.... best growth at 15° C, 20° C, 25° O, , Consistency, slimy, viscid, unchanged. 837° CG, 40° C, 50° C, 60° CG, einen ce oe dag ee Ss ed blued, greened. Maximum temperature for growth........ Cc. Shining oe 8. Litmus Milk, Bee? se Minimum temperature for growth........- Cc. > Dull ib Acid, alkaline, acid then alkaline, no change. 10. Killed readily by drying: resistant to drying. 8 - ie er ei 92, taduation. gartal. stow re: 11. Per cent killed by freezing (salt and crushed ice g 3 _Wrinkled ee 9. Gelatin Colonies. ; OY Haul airy cee... s eens . 5 Chromogenic 15 ome Growth, slow, rapid. F 3 12. Sunlight: Exposure on ice in thinly sown agar Lassa a Gael Pe te ane eee plates: one-half plate covered (time 15 min- 3 ro = ik Je Elevation, fat, effuse, raised, conver, pulvinate, utes), sensitive, not sensitive. = o, roteus-like a crateriform (liquefying). Ti oe f z ee — Edge, Bibi Suaaiote lobate, erose, lacerate, Per Mere killed my] oO | Rhizoid jimbriate, filamentous, floccose, curled. 18. Acids produced ............6. . rol B Filamentous as Liquefaction, cup, saucer, spreading. 14. Alkalies produced ............+ = o = poe Seer Cn ti emmaeatara ine OAteDnClseee sakes) oasis. +s ‘ a Curled = y r , temperature. ......0+-+ : : : A Ww roo, Lu pets , irreguigr, amebdoid, 16. Ferments; pepsin, trypsin, diastase, invertase, ee @®\| Surface-growth be ont myceliow, pnp id. : ; pectase, cytase, tyrosinase, oxidase, perocidase, a | 2s = ag eee aeahs ¥ 6 Piaget, raat: lipase, catalase, glucase, galactase, lab, etc..... Fae Bail Needle growth 7 Elevation, flat, effuse, raised, convex, pulvinate, rane see eeeeeeneesess Es os cee S_, | Moderate, absent | — idee, cance undulate, lobate, erose, lacerate, se Eg —, 1g Abundant ee fimbriate, floccose, curled. 18. Effect of germicides: S Tinculoredt ie Internal structure, amorphous, finelv-, coarsely- 5 granular, grumose, filamentous, floccose, curled, sls Stirchidectroved 11. Starch Jelly. y 8 les z Growth, scanty, copious. Biles Ze Grows at 37° C. + Diastasic action, absent, feeble, profound. 2|&|z° a i S)/2/Se Grows, in Cohn’s Sol. Mitcate salty Gk rmi’s Solution) Substance Method used. a| Slo i —— : 12. Silicate Jelly ermi’s Solu A a an 5 F = ‘ Growth copious, scanty, absent. 2 Ey oo wa Grows in Se ne Medium ne isiaislele se eeeeeee ale|s ae | Gelatin (4) qe 13, Cohn’s Solution. &|/8/S lee uy pista aes : Grawih; copious, scanty, absent. Sle | Mm ioe a Blood-serum Ve Medium fluorescent, non-fluorescent. s B/So | Bassin cers 14. Uschinsky’s ce eareecgal Brent = 9 ies |_ as Growth copious, scanty, . . 5s = lee = Vluid viscid, not viscid. = Agar, mannan 15. Sodium Chloride in Bouillon. be = a S2 _——s a = | Acid curd Per cent inhibiting growth.....-.... lg rat | = a prs C 16. Growth ae ace. See Chloroform, Pea te ey :" - _| a= Ronvciaurd strained, feeble, a “i ‘ DS ee : 17. Nitrogen. | Obtained from peptone, asparagin, es ES | | rm Casein peptozized glycocoll, urea, ammonia salts, nitrogen. | ie ae resus = 18. Best media for long-continued growth......--+++* Me = | = < Indol (5) +2 Se eee een tw niefaleferns d/alnislersiscmbianie]~'= s'ei0'¥ (58 ——. —— a ry me Hydrogen sulphide 19. Quick tests for differential purposes. ...-++-+--+++ oe — ——|—— - —_— > = _ Ammonia (©) | éicjn ar RO STEN cara ota alw ete mialave qa 'elae = MMs emery + 0% == — —— | — |——_ —_ = 4 Nitrates reduced (5) Rn ee So ec Coe SOE aio nT ana Hiageeenent ‘6 EMICAL FEATURES. III. PHYSICAL AND rocees IV. PATHOGENICITY. eiisaads o|9 eS cies hn aS oo r a * Animal pathogen, epizvon Fermentation-Tubes con- | 4) &) 0) oF) 4 Insects, crustaceans, fishes, reptiles, birds, mice,rats, ear mere Mah ake ata alge B35 S/2/ 8/5 guinea pigs, rabbits, dogs, cats, sheep, goats, Plant pathogen, epiphyte aes 3S = alg a S cattle, horses, MONREYS, MAN. ...-6.. eee sense Soil Aya} =| Re 2. Pathogenic to Plants : (o| Milk =? Gas production, in per cent. : RR RP ES ig oO a Rem ea iia aa B Fresh Sale ome —- + ( as.) No ean DIDIER oe oo Re ee = Salt water Y = sae | Fe at ei 8. Toxins, soluble, endotoxins, | S | Sewage y +t. ‘eta bes ae ssi | —|—|—- *, % eathgiged pe ara 1 ine) "Tron bacterium Amount ofacid producedi d. . Immunity bactericidal. Zz : a ee ei ; ats = “sea fac id 6. Immunity non-bactericidal. | Sulphur bacterium dD 2d. < d | 2 ke Me Pa Wl 7. Loss of virulence on culture media: prompt, sy Sea: * 4d. gradual, not observed in.------+ Witten eee months.

eo.

DESCRIPTIVE CHART—SOCIETY OF AMERICAN

Prepared by F. D. CHESTER

F P. GORHAM ERWIN F. SMITH

ENDORSED BY THE SOCIETY FOR GENERAL USE AT THE ANNUAL MEETING, DEC. 31, 1907.

GLOSSARY OF TERMS.

AGAR HANGING BLOCK, a small block of nutrient agar cut from a poured plate, and placed on a cover-glass, the surface next the glass having been first touched with a loop from a young fluid culture or with a dilution from the same. It is examined upside down, the same as a hanging drop.

AMEBOID, assuming various shapes like an ameba.

AMORPHOUS, without visible differentiation in structure.

ARBORESCENT. a branched, tree-like growth.

BEADED. in stab or stroke, disjointed or semi-confuent colonies along the line of inoculation,

BRIEF, a few days, a week.

BRITTLE, growth dry, friable under the platinum needle.

BULLATE, growth rising in convex prominences, like a blistered surface.

BUTYROUS, growth of a butter-like consistency.

CHAINS,

Short chains, composed of 2 to 8 elements. Long chains, composed of more than 8 elements.

CILIATE, having fine, hair-like extensions like cilia.

CLOUDY, said of fluid cultures which do not contain pseudozoogloeae. =

COAGULATION, the separation of casein from whey in milk. This may take place quickly or slowly, and as the result either of the formation of an acid or of a lab ferment.

CONTOURED, an irregular. smoothly undulating surface, like that of a relief map.

CONVEX. surface the segment of a circle, but flattened.

COPROPHYL, dung bacteria.

CORIACEOUS, growth tough, leathery, not platinum needle.

CRATERIFORM, round, depressed, due to the liquefaction of the medium.

CRETACEOUS. growth opaque and white, chalky.

CURLED, composed of parallel chains in wavy strands, as in anthrax colonies.

DIASTASIC ACTION, Same as DIASTATIC, conversion of starch into water-soluble substances by diastase.

ECHINULATE, in agar stroke a growth along line of inocula- tion. with toothed or pointed margins; in stab cultures growth beset with pointed outgrowths.

EFFUSE, growth thin, veily. unusually spreading.

ENTIRE, smooth, having a margin destitute of teeth or notches.

EROSE, border irregularly toothed.

FILAMENTOUS, growth composed of long, irregularly placed or interwoven filaments.

FILIFORM, in stroke or stab cultures a uniform growth along line of inoculation,

FIMBRIATE, border fringed with slender processes, larger than filaments.

FLOCCOSE, growth composed of short curved chains. variously oriented.

FLOCCULENT, said of fluids which contain psevdozcogloeac, i. e.. small adherent masses of bacteria of various shapes and floating in the culture fluid.

FLUORESCENT, having one color by transmitted light and another by reflected light.

GRAM’S STAIN, a method of differential bleaching after gentian violet, methyl violet, etc. The + mark is to be given only when the bacteria are deep blue or remain blue after counterstaining with Bismark brown.

GRUMOSE, clotted.

INFUNDIBULIFORM, form of a funnel or inverted cone,

IRIDESCENT, like mother-of-pearl. The effect of very thin films.

yielding to the

LACERATE, having the margin cut into irregular segments as if torn.

LOBATE, border deeply undulate, producing lobes (see undulate.)

LONG, many weeks, or months.

MAXIMUM TEMPERATURE, temperature above which growth does not take place,

MEDIUM, several weeks.

MEMBRANOUS, growth thin, coherent. like a membrane.

MINIMUM TEMPERATURE, temperature below which growth does not take place.

MYCELIOID. colonies having the radiately filamentous appear- ance of mold colonies,

NAPIFORM, liquefaction with the form of a turnip.

NITROGEN REQUIREMENTS, the necessary nitrcgenous focd. This is determined by adding to mitrogen-free media the nitrogen compound to be tested.

OPALESCENT, resembling the color of an opal.

OPTIMUM TEMPERATURE, temperature at which growth is most rapid.

PELLICLE, in fluid bacterial growth either forming a continuous or an interrupted sheet over the fluid.

PEPTONIZED, said of curds dissolved by trypsin.

PERSISTENT, many weeks, or months,

PLUMOSE, a fleecy or feathery growth. ;

PSEUDOZOOGLOEAE, clumps of bacteria, not dissolving readily in water, arising from imperfect separation, or more or less fusion of the components, but not having the degree of compactness and gelatinization seen in zoogloeae.

PULVINATE, in the form of a cushion, decidedly convex.

PUNCTIFORM, very minute colonies, at the limit of natural vision.

RAISED, growth thick, with abrupt or terraced edges.

RHIZOID, growth of an ifregular branched or root-lfke character, as in B. mycoides.

RING. Same as RIM, growth at the upper margin of a liquid culture, adhering more or less closely to the glass.

REPAND. wrinkled.

RAPID, Developing in 24 to 48 hours.

SACCATE, liquefaction the shape of an elongated sack, tubular, eylindrical,

SCUM, floating islands of bacteria, an interrupted pellicle or bacterial membrane. 3

SLOW. requiring 5 or 6 days or more for development.

SHORT. applied to time, a few days, a week.

SPORANGIA, cells containing endospores.

SPREADING, growth extending much beyond the line of inoculation, i. e., several millimeters or more.

STRATIFORM, liauefying to the walls of the tube at the top and then proceeding downwards horizontally.

THERMAL DEATH-POINT, the degree of heat required to kill young fluid cultures of an organism exposed for 10 minutes (in thin-walled test tubes of a diameter not exceeding 20 mm.) in the thermal water-bath. The water must be kept agitated so that the temperature shall be uniform during the exposure. !

TRANSIENT. a few days.

TURBID. cloudy’ with floceulence.

UMBONATE, having a button-like. raised center.

UNDULATE, border wayy. with shallow sinuses.

VERRUCOSE, growth wart-like. with wart-like prominences.

VERMIFORM-CONTOURED, growth like a mass of worms, or intestinal coils.

VILLOUS, growth beset with hair-like extensions.

VISCID, growth follows the needle when touched and withdrawn, sediment on shaking rises as a coherent swirl.

ZOOGLOEAE, firm gelatinous masses of bacteria, one of the most typical examples of which is the Streptococcus mesenter- ioides of sugar vats (Leuconostoc mesenterioides), the bac- terial chains being surrounded by an enormously thickened firm covering, inside of which there may be one or many groups of the bacteria.

floceulent particles; cloudy plus

BACTERIOLOGISTS

Committee on Methods of Identification of Bacterial Species,

NOTES.

(1) For decimal system of group numbers see Table 1. This will be found useful as a quick method of showing close rela- tionships inside the genus, but is not a sufficient characterization of any organism.

(2) The morphological characters shall be determined and described from growths obtained upon at least one solid medium (nutrient agar) and in at least one liquid medium (nutrient broth). Growths at 87° C shall be in general not older than 24 to 48 hours, and growths at 20° C not older than 48 to 72 hours. To secure uniformity in cultures, in all cases preliminary cultiva- tion shall be practiced as described in the revised Report of the Committee on Standard Methods of the Laboratory Section of the American Public Health Association, 1905.

(3) The observation of cultural and bio-chemical features shall cover a period of at least 15 days and frequently longer, and shall be made according to the revised Standard Methods above referred to. All media shall be made according to the same Standard Methods.

(4) Gelatin stab cultures shall be held for 6 weeks to deter- mine liquefaction,

(5) Ammonia and indol tests shall be made at end of 10th day, nitrite tests at end of 5th day.

(6) Titrate with Jy NaOH, using phenolphthalein as an indicator: make titrations at same times from blank. The difference gives the amount of acid produced.

The titration should be done after boiling to drive off any Co2 present in the culture.

(7) Generic nomenclature shall begin with the year 1872

(Cohn’s first important paper). Species nomenclature shall begin with the year 1880 (Koch’s discovery of the poured plate method for the separation of organisms). (8) Chromogensis shall be recorded in standard color terms. TABLE I.

A NUMERICAL SYSTEM OF RECORDING THE SALIENT CHARACTERS OF AN ORGANISM. (GROUP NUMBER.)

100. Endospores produced 200. Endospores not produced 10, - Aerobie (Strict) 20. Facultative anaerobic 30. Anaerobic (Strict) aw Gelatin liquefied 2 Gelatin not liquefied 0.1 Acid and gas from dextrose 0.2 Acid withoyt gas from dextrose 0.3 No acid from dextrose 0.4 No growth with dextrose 01 Acid and gas from lactose -02 Acid without gas from lactose *

03 No acid from lactose

04 No growth with lactose

-001 Acid and gas from saccharose -002 Acid without gas from saecharose 003 No acid from saccharose

-004 No growth with saccharose

0001 Nitrates reduced with evolution of gas -0002 Nitrates not reduced

.0003 Nitrates reduced without gas formation -00001 Fluorescent

.00002 Violet chromogens

-00008 Blue =

.00004 Green Rf

00005 Yellow as

00006 Orange se

00007 Red re

-00008 Brown

.00009 Pink g

.00000 Non-chromogenic

-000001 Diastasic action on potato starch, strong -000002 Diastasic action on potato starch, feeble -000003 Diastasic action on potato starch, absent -0000001 Acid and gas from glycerine

0000002 Acid without gas from glycerine .0000003 No acid from glycerine

-0000004 No growth with glycerine

The genus according to the system of Migula is given its proper symbol which precedes the number thus: (7)

BACILLUS COLI (Esch.) Mig. becomes B. 222.11110: BACILLUS ALCALIGENES Petr. “-B, ——-212.833102) PSEUDOMONAS CAMPESTRIS (Pam. ) Sm. “ Ps. 211.333151

BACTERIUM SUICIDA Mig. * Bact. 222.23!

——_-

.

BIBLIOGRAPHY

1. Microorganisms in Water. Percy and G C. Frankland. 1894. p. 105. 2. Water Supplies. Savage. 1906. Chapter IX. 3. Elements of Water Bacteriology. Prescott and Winslow. 1913. p. 23. 4. Examination of Water and Water Supplies. 1912. 5. Notes on the Distribution of B. coli. Flint. Jour. jmer. Med. Assn. 1896. Vol. 26. p. 410. 6. Zur Lehre uber das Bacterium coli commine. Belitzee. Rev. Jahresbericht uber die Fortschritte in der Lehre den pathogenen ilikroarganismen. Vol. 25. p.326. 7.% Notes on Normal Intestinal Bacilli of the Horse and of Certain Other Domesticated animals. Dyer and Kieth. Tech. Quarterly. Vol. 6. p. 256. 7.& The Occurrence of Organisms of Sanitary Significance on Grains. Prescott. Biol. Studies of Pupils of W. T. Sedgwick. 1906. 8. Organisms on the Surface of Grains with Special Reference to B. coli. Metcalf. Oct. 6, 1905. Science. Vol. 22. 9. Occurrence on Grain of Organisms Resembling the B. coli communis. HE. G. Smith.

Science. Vol. 21. p. 710.

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Kritische und experimentelle Beitrage zur hygienischen Beurteilung des Wassers. Kruse.

Zeit. f. Hyg. Vol. 27. p. l. Ueber die typhus ahnlichen Bakterien des Strassburger Wasserleitungswasser. Beckmann.

Arch. f. Exp. path. und Pharm. Vol. 33. p. 466. Bakterium coli commune. Maroni.

arch. per le Science medich. Vol. 22. p. 261. Der Befund des Bakterium coli im Wasser und das Teirexper- iment sind keine brauohbare Hulfmittels fur die Hygieniache Beurteilung des Wassers. Weissenfeld.

Zeit. £f. Hyg. Vol. 35. p. 78. The Distribution of Bacterium coli commune. Chick.

The Thompson Yates Lab. Reports. Report 3. p. l. The Colon Bacillus in Ground Waters. Horton.

Public Health. Vol. 28. p. 419.

The Sand Filtration and Purification of Chalk Waters. Nankivell. 1911. Jour. of Hyg. Vol. ll. p. 235. Experiment Relating to Well Contamination at Quitman, Ga. Mac Callie. Water Supply Paper. No. 110. p. 145.

Use of Flourescein in Tracing Waters. R. B. Dole. Water Supply Paper. No. 160. p. 73.

Sur l'emploi matieres coulerantes pour la recherche

de l'origin des sources et des eaux d’infiltration. Trillat. Compt. Rend. 1699. Vol. 128. p. 698.

Contribution 41° étude des eaux souterraine. Marboutin.

Compt. Rend. 1901. Vol. 132. p. 3686.

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Bakteriologische Untersuchungen des Trinkwassers der

Is the Colon Bacillus a Normal Inhabitant of the Intes- tine of Fishes. Amyot. Public Health. Vol. 27. p. 400. Isolation of B. coli from the alimentary tract of fish and the Significance thereof. Geo. 4. Johnson. Jour. Inf. Dis. Vol. 1. p. 348. 4 Comparison of B. coli communis from Different Species of Animals. Moore and Wright. | Jour. Bost. Med. Soc. Vol. 4. p. 175. Distribution of B. coli in Nature. Eyre, J. W. H. Lancet (1904). Vol. 1. p. 648.

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