AND DIFFUSION IN ENDO AGAR THEORIES FOR COLOR PHENOMENA IN ENDO AGAR Thereason for this color phenomenon has been explained in various ways by different workers, Endo (1904) explains

COLOR DIFFUSION IN ENDO AGAR

ELIZABETH F. GENUNG AND LUCY E. THOMPSON

Department of Botany, Smith College, Northampton, Massachusetts

Received for publication, December 9, 1926

Bacteriologists have long recognized the need for a reliabletest to detect the presence of colon organisms and to differentiatebetween the various species of that group. In 1904 Endo pub-lished a test which has been recognized ever since as a valuableand a desirable one. It has been widely used, especially in thiscountry, for studying members of the colon group of bacteria.This medium contains an indicator in the fonn of decolorized

basic fuchsin. When Bact. coli is grown on Endo agar it bringsabout a reaction which leads to a restoration of the color of theftichsin in the colony while other bacteria have no such effect ondecolorized fuchsin. This is an excellent and efficient methodfor detecting Bact. coli in contaminated materials, but it hascertain disadvantages which have not, as yet, been entirelyovercome.The method for making this medium is described by Endo as

follows:

Neutral 3 per cent agar broth............................ 1000 cc.Lactose C.P............................ 10 gramsAlcoholic fuchsin............................ 5.0 cc.10 per cent sodium sulphite solution........................ 2.5 cc.10 per cent sodium carbonate solution...................... 10.0 cc.

Cook the agar broth well, filter, neutralize and then add 10 cc. of a10 per cent solution of sodium carbonate to make it alkaline. Thenadd the lactose and fuchsin solution. This will make the medium red.Now add the sodium sulphite solution which will gradually decolorizethe medium so that it will be entirely colorless when the agar is cold.After the medium has been put into tubes or flasks, it is sterilized forthirty minutes in the autoclave.

139

on May 11, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

140 ELIZABETH F. GENUNG AND LUCY E. THOMPSON

Precautions

1. The lactose must be chemically pure, for the commercial lactosecontains ordinary cane sugar with which the typhoid bacillus will makeacid and it will be very hard to distinguish it from the colon bacterium.

2. The sodium sulphite solution must be stored in a tightly stopperedflask or made up fresh.

3. The alcoholic fuchsin must be filtered beforehand.4. The medium must be stored in the dark, otherwise by exposure to

light the red color returns. When the medium is used, it is melted andpoured into sterile plates and left standing uncovered in a dust freeplace until the plates harden. After they have hardened the plateswill be colorless and transparent.

GROUPS OF COLON BACTERIA

There are four distinct classes of organisms belonging to thecolon group which are classified according to their ability toferment ordinary laboratory sugars:

a. Those organisms which ferment glucose and lactose with theproduction of both acid and gas in each.

b. Those which produce acid and gas in glucose, but which do notferment lactose.

c. Those which produce acid only in glucose and lactose.d. Those which produce acid only in glucose and do not ferment

lactose.

The individual species within these groups ferment various othersugars, but for the purpose of this article these reactions are notessential.

Because of the varying ability of these organisms to fermentlactose, it is possible to detect members of the groups outlinedabove on Endo agar. Those which ferment lactose with theproduction of acid and gas are able to restore the color of thedecolorized fuchsin, while those which produce acid only inlactose restore but a faint pink color, and the colonies of thoseorganisms which do not ferment lactose remain colorless.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


THEORIES FOR COLOR PHENOMENA IN ENDO AGAR

The reason for this color phenomenon has been explained invarious ways by different workers, Endo (1904) explains it asfollows:

The chemistry of the color change in the medium is caused by thefollowing means: Fuchsin contains essential acid salts of rosanilin,C2H19N2HCl. Rosanilin is a colorless, so-called leucko-base which withdifferent acids, such as lactic acid, hydrochloric acid, etc. produces ared color. The acid compound of red rosanilin salts, is easily reducedthrough a reducing agent such as sodium sulphite. This, the colorlessrosanilin combines with, through the production of acid by the colonbacillus, and the medium becomes a bright red.

Harding and Ostenberg (1912) commenting on Endo's explana-tion of the chemical change causing the restoration of color saythat it offers a plausible explanation which seems to have beenaccepted, but it apparently was not based on experimentalinvestigation. A summary of the explanation of the colorphenomenon, by these authors, is, that the colorless solutionobtained by adding fuchsin to sodium sulphite or sodium bisul-phite or sodium dioxide in solution is used in chemical analysisfor the quantitative determination of aldehydes. This solutionis known as Schiff's aldehyde reagent. Acids do not give anycolor test with this reagent and, if present in sufficient quantity,prevent aldehydes from giving it. These authors quote Nietskias giving the following reaction between fuchsin, sodium sulphiteand aldehydes: "Rosanilin and pararosanilin form with sulfurousacid and the alkali bisulfites colorless, easily decomposed' com-pounds. By the action of aldehydes upon these bodies peculiarviolet dyes are formed."Robinson and Rettger (1916) have still another theory. They

explain the reddening of the colon colonies by the presence inthe medium of decomposition products of lactose, such as lacticacid and some acetic acid. These acids cause oxidation of thecolorless leuco-base to the fuchsin color product. The decolori-zation, which takes place if the plate is allowed to stand long

141


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


enough, is due to such alkalis as amnmonia and amines, which areformed from the peptone and the beef extract.DeBord (1917) performed some experiments to test the effect

of acid alone, combinations of acid and formaldehyde, and form-aldehyde alone on decolorized basic fuchsins as well as on Endoagar. He thinks the reaction is an acid-aldehyde reaction andthat the presence of an aldehyde is necessary to it.

MODIFICATIONS OF ENDO 'S METHOD

This medium has become generally known as Endo's mediumor Endo agar and has been in general use in bacteriologicallaboratories since its discovery. Efforts have been made fromtime to time to modify it, in order to correct some of the defectswhich lead to a lack of uniformity of results among differentworkers. Endo added 10 grams of sodium carbonate "to makethe medium alkaline." This has been found to be too empiricala method of adjusting the reaction. Accordingly, careful titra-tion of the amount of acid with phenolphthalein as an indicatorand the use of NaOH to adjust the reaction to the desired alka-linity has been substituted. A still more accurate method is todetermine the hydrogen ion concentration and adjust the reactionby this method.

In spite of a more carefully adjusted alkalinity, there is stillsome difficulty in securing a satisfactory medium. The chieftrouble has probably been found with the color diffusion. Endoagar, prepared for use in the laboratory, develops a decidedcolor on standing. Endo encountered this difficulty, and cau-tions that the medium must be kept in the dark as exposure tolight causes a return of color. Later it was found that even slightamounts of acid caused the return of color in a sterile medium ormight even prevent the complete decolorization of the agar.The importance of an alkaline reaction is thus emphasized.Robinson and Rettger (1916) state that the final reaction and

the use of pure reagents, especially the lactose and sulphites are themost important factors in securing a satisfactory Endo agar.They recommend the use of C.P. anhydrous sodium bisulphite


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


as a decolorizing agent, since poor results have been obtainedwith both crystalline and anhydrous sodium sulphite.

Levine (1921) has tried to solve the problem by substituting adipotassium salt for the beef extract, which he believes simpli-fies the process and makes for a better differentiation betweenBact. coli and Bact. aerogenes. He does not adjust the reactionof this medium, but states that "The diffusion of color due tothe production of acid is very rapid."Genung (1924) suggests that the restoration of color may be

due to the pepton, having found two different brands of peptonresulted in marked differences in the color on sterile Endo agarplates incubated for twenty-four hours.

Harris (1925) finds that the hydrogen ion concentration andthe brand of pepton, both have definite effects on the restorationof color in uninoculated plates.

IMPORTANCE OF A CLEAR CUT REACTION IN DETECTING THE KIND

OF ORGANISMS PRESENT ON THIS MEDIUM

There is a marked difference in the degree of color producedby different species of the colon group of bacteria on this medium.Bact. coli colonies produce a deep, iridescent, fuchsin red, whilethe Bact. aerogenes colonies are a rose pink. Bact. dysenteriae andBact. para-typhosum A have pale pink colonies, while Bact. ty-phosum and Bact. typhosum B colonies remain colorless. If themedium becomes highly colored, it diffuses into the growth sothat typhoid colonies, for exampIe, may become pink and re-semble the colonies of dysentery or of Bact. aerogenes. It isimportant, if careful differentiation is desired, to have a mediumwhich will remain colorless, except in the region of the colonies.For several years the writer experienced considerable difficulty

in securing a colorless Endo agar for class room work. Variousbrands of both crystalline and anhydrous sodium sulphite weretried and care was taken to have the reaction decidedly alkaline,but these precautions did not eliminate the difficulty. In con-nection with some laboratory experiments, Endo agar had beenmade up with two different brands of pepton. In testing thesterility of this agar by twenty-four hours incubation of the

143


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


plates, it was found that the plates made from the "Difco" brandof pepton, ordinarily used in this work, were a decided pink;while those made from Witte's pepton remained colorless. Allthe plates were sterile. This led to further tests and a seriesof experiments were undertaken to study the effect of differentbrands of pepton on the color diffusion.

MODIFICATIONS NECESSARY TO FACILITATE THESE TESTS

Several modifications have been suggested by different workersin the preparation of this medium. None of these materiallyaffects the composition as outlined in the original formula, butthey have primarily to do with facilitating processes of prepara-tion. In choosing the formula for these experiments, it wasthought best to use the method recommended by the Committeeon Standard Methods of Water Analysis (1923) since that is,probably, the one most generally employed in public healthlaboratories.From the beginning it was found necessary to alter this method

somewhat, not only to facilitate the preparation, but to elimi-nate all possible chances of contamination. The method finallyworked out and used in these experiments is given in some detailhere, because it has frequently been charged that the methodused in preparation accounts for the differences in results ob-tained by different workers. The medim used in these testswas as follows:

Beef extract.......... 5 gramsPepton.......... 10 gramsDistilled water.......... 1000 cc.Agar.......... 30 grams

The beef extract and pepton were dissolved in water and theagar added. The mixture was brought to a boil and autoclavedat fifteen pounds pressure for twenty minutes. At the sametime the filters and flasks were sterilized. After removing fromthe autoclave the reaction was adjusted, 10 grams of lactose wereadded and the medium filtered into flasks. It was again auto-claved at ten pounds pressure for fifteen minutes.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


When ready for use, to each 100 cc. of the agar was added 10cc. of a 2.5 per cent solution of sodium sulphite in which 0.5 cc.of an alcoholic solution of basic fuchsin had been decolorized.It was immnediately poured into plates and allowed to hardenbefore replacing the covers.The method which was used for titrating the reaction was the

colorimetric one recommended by Jordan (1924). To 4 cc.of heated distilled water was added 1 cc. of the agar and fivedrops of Brom Thymol Blue as indicator. The medium wascarefully adjusted to the desired hydrogen ion concentration andthe reaction rechecked. This method was found to be mostsatisfactory as the highly diluted agar reduces the error causedby the masking of the color of the indicator in a less dilute medium.It also checked with the other methods in accuracy.

TESTS WITH DIFFERENT BRANDS OF PEPTON

The first factor which was tested was the pepton factor. Sixdifferent brands of pepton on the market for culture media workwere secured. They were Witte's, Armour's, Difco, Fairchild's,Parke-Davis' and Merck's. The latter has been discontinuedsince these tests were begun. Endo agar was prepared usingthese peptons and plates were poured. They were allowed toharden and then divided into three groups, one set was inocu-lated with Bact. coli and one with Bact. aerogenes while the thirdwas not inoculated. Since exposure to light has always beenthought to have an effect on the production of color, the experi-ment included this factor. Uninoculated plates were left exposedto the daylight, while others were exposed to the light of anordinary electric light bulb. The remainder were kept in thedark. It was found to be inadvisable to inoculate the daylightplates as the colonies never grew well. Table 1 shows the resultsof this experiment. The work was carefully rechecked manytimes at varying intervals and the results were found to beuniform.There is the least return of color in the plates containing

Witte's pepton agar while Armour and Merck pepton agars showthe greatest amount of color. The amount of color at the end

145


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


of twenty-four hours incubation was comparatively small evenin those plates which were exposed to light, when plates madefrom the same pepton were compared. After being incubatedfor an additional twenty-four hours, there was still practicallyno difference in the relative amounts of color in plates of agarmade from the same brand of pepton, whether the plateswere incubated in the dark, in artificial light or exposed to

TABLE 1

The effect of peptones and light on restoration of color in endo agar

DARK INCUBATOR LIGHT INCUBATOR

PUPTONU DDAYLIGHTUXPOSURN Inoeu- Not Inocu- Notlated linoculated lated inoculated

After twenty-four hours

Witte...........Parke-Davis.......+. + + _Fairchild....... + + + ++ ++Difco....... + 1+ + + +Merck....... ++ ++ ++ ++ +Armour. ++ ++ ++ +'+ ++

A fter forty-eight hours

Witte....... + ++ - +++ +Parke-Davis.........................++ + +++ + ++..++ +Fairchild......... ++ +++ ++ +++ ++Difco....... +++ +++ ++ ++++ +Merck....... +++ ++'++ ++ ++++ +Armour. ++ ++++ ++ ++++ +

- no color visible; + faint pink tinge; ++ distinct pink tinge; +++ wholeplate rose pink; ++++ whole plate deep rose red.

daylight. The return of color after the first twenty-four hoursincubation was more marked and took place more rapidly thanduring the first period.The different brands of pepton were tested for their pepton

content by a chemist. The results of these tests are shown intable 2. This table shows their compaxative solubility and thecolor of the solutions. According to the biuret test these pep-tones vary considerably in their actual pepton content. An


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

COLOR DIFFUSION IN ENDO AGAR 147

interesting fact may be pointed out in connection with theParke Davis product. This firn discontinued their old brandof pepton and replaced it with a new brand which they labelled,"Bacteriologic Pepton." This new product gave a negativebiuret test while the discontinued product was fairly high in

TABLE 2

Tests for Pepton ContentSolutions: 1 gram in 100 cc. of distilled water

BRAND

Witte ...............

pH

7.0

Parke-Davis........ 6.4

tParke-Davis........I 5.7

SOLUTION

Heavy sus-pension

Slight sus-pension

Slight sus-pension

Fairchild.... 5.4 Not perfect

Difco........Armour.............Merck...............

7.06.24.0

PerfectPerfect

Fine suspen-sion

COLOR OF SOLU-TION

Colorless

Very slightcolorationVery faintyellow

Deep yellow

Faint yellowFaint yelow

Orange

BIURETTEST

++

++

_ Not rose pinkof true pep-tone. Purplewith strongpink tinge.

- A large excessof 40 per centNaOH bringsout the biuretpink

++

With excess of40 per centNaOH color ispurpose-pink

+ positive test; ++ strongly positive test; - negative test.* One cubic centimeter of 1 per cent NaOH + 3 drops of 2 per cent CuSO4.t New product of the Parke-Davis Company called "Bacteriologic Pepton."

pepton content. Another fact which this test brought out was,that Witte's pepton, which seems to inhibit the return of colorto a greater extent than other brands, contains a heavy insolubleprecipitate in water which does not dissolve when the mixture isheated.

Since the reaction of the medium appears to influence theamount of color production, the agar was adjusted to various


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


reactions from pH 7.0 to 7.8. It was found that the growthof the organisms was inhibited when the range was above pH 7.6and that a pH of 7.0 was not alkaline enough, since the plateswere all highly colored. A careful test was made of the com-parative effects of pH 7.2 and 7.4 on the color. These resultsare shown in table 3. There is relatively little difference in the

TABLE 3

The relation of reaction of medium to restoration of color

pH 7.2 pH 7.4

PUPTONE Bact.Day- Dark Bact. Bact. Day- Dark Bact. coli aero-light coli aerogenes light

After twenty-four hours

Witte.................Parke-Davis........ - + + + _ +Fairchild....... _ - + +++ + + + +Difco....... + + ++ ++ + + + +Merck....... +++ + + ++ ++ + ++ ++Armour........+ + .+ +++ ++ ++ ++ ++

After forty-eight hours

Witte ............... - - ++ ++ ++ +Parke-Davis..........- + +++++++ - - ++ +Fairchild............ + + ++ ++++ + + +++ ++Difco............... + + +++++++ + + +++ ++Merck............... +++ + +++ +++ + + ++++ ++Armour............... + + +++++++ + + +++ +++

- no color visible; + faint pink tinge; ++ distinct pink tinge; +++ wholeplate rose pink; ++++ whole plate deep rose red.

amount of color production in the different pepton agars betweenthe two reactions. A pepton which does not inhibit color pro-duction has about the same depth of color in both reactions.After forty-eight hours incubation, the color is more marked inthe pH 7.2 plates. Since there was this slight difference, thereaction pH 7.4 has been used in all subsequent work.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


FUCHSIN AS A FACTOR IN COLOR DIFFUSION

During these tests it became evident that the fuchsin playedan important part in the return of color. Accordingly a carefulstudy was made of this factor. Endo (1904) stated that 5 cc.of basic fuchsin was to be added to each liter of agar. He gaveno directions as to the strength of the solution, only stipulatingthat it must be filtered. Later authors recommended a satu-rated alcoholic solution of basic fuchsin. A saturated alcoholicsolution of basic fuchsin is usually made by adding one part ofthe dry fuchsin to ten parts of 95 per cent alcohol. This mixtureis allowed to stand twenty-four hours then the supernatant fluidis filtered off and used in the Endo agar. The common practiceis to add 5 cc. of this fuchsin solution to each liter of agar, togetherwith 10 cc. of a 2.5 per cent solution of sodium sulphite todecolorize the fuchsin.

DIFFICULTIES ENCOUNTERED WITH AMERICAN FUCHSINS

When American brands of fuchsin came into the laboratory,much difficulty was encountered in decolorizing the agar by this,method. It was found to be more satisfactory to add the fuchsinto the sulphite solution before adding it to the agar. This methodresulted in the discovery that different makes of American fuch-sin did not decolorize in the sodium sulphite solution in thepreviously mentioned proportions. The fuchsin not only failedto become decolorized, but formed a thick, flocculent precipitatein many cases. Careful tests of a large number of fuchsins weremade, in order to discover their ability to become decolorized inthe sodium sulphite solution. The results of these tests havealready been published (Genung, 1926a) so that it is necessaryto give only a brief summary here.

It was found that different lots of fuchsin, even from the samefirn, vary in their solubility in alcohol (Conn, 1923) and thatfurther dilution is often necessary before they will be decolorizedby the sodium sulphite solution. A scheme was devised wherebyeach new lot of fuchsin could be tested for this property. Aseries of dilutions of the saturated, alcoholic solution of the

149


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


fuchsin were made and each was tested in a sodium sulphitesolution of the same strength and in the same proportions usedin Endo agar. The lowest dilution which was decolorized bythe sodium sulphite solution was used in the agar. This wasfurther tested by inoculating plates made with this solution ofthe fuchsin with known organisms. Bact. coli and Bact. aerogeneswere used and if these organisms produced characteristic reac-tions on the agar, the dilution was considered satisfactory. Itwas also found by these tests that some fuchsins were neversatisfactory. They either would produce too much color in lowdilutions or too little color in higher dilutions or sometimes theywere not sufficiently decolorized by the sodium sulphite solu-tion, even in high dilutions, to make it advisable to use them forthis work.When carefully tested and properly diluted fuchsin was used

there was the same general variation in color return in the platesof agar made from the different peptones. These varia-tions remained uniform throughout the experiments. If a pep-ton did not inhibit color production in the agar, its effect wasonly accentuated when too strong a solution of fuchsin was used;while a pepton which inhibited the color production had a deepercolor in the stronger fuchsin solutions. It would seem that thefuchsin factor is an important one, but at the same time, onecapable of solution.

THE EFFECT OF LIGHT ON COLOR PRODUCTION

As was noted in a previous paragraph, experiments werecarried on to test the effect of exposure to light on the return ofcolor in Endo agar. Various tests of this factor were made.In each batch of plates a certain percentage were exposed toboth daylight and artificial light. Tubes and flasks of the agarwere also exposed to light and compared with duplicates keptin the dark.The results of these tests with the plates are recorded in table

1. They seem to indicate that light as a factor in the return ofcolor in Endo agar is fairly negligible. When exposed to artificiallight or daylight, the plates developed very little more color than


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


those kept for the same length of time in the dark. The differentpeptones showed characteristic reactions in either case. Thosewhere the color diffusion was marked developed the same degreeof color in the dark as in the light. Those peptones whichinhibited color production developed but little color in the light.After twenty-four to thirty-six hours incubation, the return ofcolor was more rapid in the light, but they all ultimately becamea deep, dark red at the end of forty-eight hours. Artificial lighthad less effect on the rapidity of color diffusion than daylight.This fact is rather interesting, since the workers in one laboratory,known to the writer felt they could not use Endo agar becausetheir incubator was heated with electric light bulbs. Agartreated in the same way in larger masses, such as tubes and flasks,showed even less effect of light. The return of color was moregradual than in the plates, but it seemed to be uniform in eithercondition.

ORGANISMS USED TO TEST THE EFFICIENCY OF THE MEDIUM

In all these experiments, two organisms were used to test theeffects of these different factors on the agar. Bact. coli and Bact.aerogenes were chosen because they produce definite, but differentreactions on Endo agar. Bact. coli gives a characteristic, iri-descent, fuchsin red colony; while Bact. aerogenes produces a rosepink color in the growth. Before completing the test of any onefactor or combination of factors, inoculations were made withother organisms to compare their reactions with those ordinarilyused. For these further tests Bact. typhosum, Bact. paratypho-sum A, and Bact. paratyphosum B were used.The results of the tests with these organisms showed that where

the medium developed a deep red color, it would often diffuse intothe colony and cause a normally white colony to become a rosepink.

MODIFICATIONS OF ENDO 'S MEDIUM SUGGESTED BY DIFFERENT

EXPERIMENTERS

There have been several attempts to alter the composition ofEndo's Medium in order to improve it and to obtain more un-

151


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

ELIZABETH F. GENUNG AND LUCY E. THOMPSON

form results. Robinson and Rettger (1916) found that sodiumbi-sulphite used as a decolorizing agent, gave more satisfactoryresults than sodium sulphite and they recommended its adoption.Levine (1921) finds that the substitution of dipotassium phos-phate in place of the beef extract gives better results, especiallyin the differentiation between certain species of the colon groupof bacteria.Some workers use 1.5 to 2 per cent agar instead of 3 per cent

agar. Levine advocates 2 per cent agar in his method. Certainmanufacturers are putting a dehydrated Endo agar on themarket, the exact ingredients of which are not always known tothe laboratory worker. Since all these changes and modificationsmay enter into the problem of color production, they were studiedand compared with the standard method.

In working with the method suggested by Robinson and Rett-ger (1916), it was found that when sodium bisulphite-fuchsinsolution was added to the agar and the plates inoculated, nogrowth appeared. The agar was apparently too acid for bacteriato grow since sodium bisulphite is highly acid. Tests were madeto ascertain how best to overcome this difficulty and it was foundthat by adjusting the reaction of the agar to pH 10.0 beforeadding the sodium bisulphite solution, the final reaction wouldbe about pH 6.8. Plates were made from this agar but the growthwas very scanty or, in some cases, no growth at all appeared.Several failures to use this method successfully resulted in its,being discarded for these experiments.

Levine's method was carefully tested. He states that it isnot necessary to adjust the reaction, but in these tests plates weremade from unadjusted medium and from the medium adjustedto pH 7.2. The reaction of the agar made from the differentpeptones varied considerably, ranging from pH 6.4 to 6.8. Whenplates of the Endo agar, containing these peptones and madeaccording to Levine's formula, were inoculated with Bact. coliand Bact. aerogenes, the results were interesting. In none of theplates did the Bact. coli colonies show the characteristic fuchsinred, on the contrary all the colonies were a deep rose red; thoseof Bact. coli being a trifle deeper color than those of Bad. aero-

152


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


genes. In the plates adjusted to pH 7.2, the colonies were afainter pink or, in some cases, there was no color at all. Therewas no return of color in the agar in any of the plates at the endof twenty-four hours incubation. This did not seem to be a goodmedium for differentiating between Bact. coli and Bact. aerogenes.The use of dehydrated Endo agar and of the standard Endo

agar containing only 1.5 per cent agar were tried but the colorin both cases was. very marked. Neither method seemed tooffer a solution to the problem.

In a previous article (Genung, 1926b), the writer has attemptedto show that the hydrogen ion concentration of the pepton has adirect effect on the return of color in the fuchsin-sulphite solution.The different brands of peptones, as will be noted in table 2,differ in their acidity, ranging from a fairly high acidity in thecase of Merck's to neutrality in Difco's and Witte's peptones.When the decolorized fuchsin was added to these pepton solu-tions, the color returned immediately in those which had an acidreaction and did not return at all in the neutral solutions. Afterall the solutions of pepton were adjusted to pH 7.0, no colorreturned upon the addition of the fuchsin-sulphite solution. Theaddition of a very nute amount of a weak acid solution and asmall quantity of formalin restored the fuchsin color completely.This bears out the theory suggested by DeBord (1917) thatthe return of color in the colonies of the colon group on Endo agaris an acid-aldehyde reaction and not due to the presence of lacticacid alone.

Since the presence of very small amounts of acid and aldehydeaffect the return of color to a marked extent, and since the pep-tones vary considerably in their actual pepton content, it isinteresting to speculate as to their true composition and to ques-tion whether the presence of simple protein and carbohydratecompounds may not be, to some extent, responsible for the returnof color in Endo agar. It is possible that there may be somesignificance in the fact that those brands of peptones which givepositive biuret tests are the most nearly neutral in their reactionand have the least diffusion of color in the agar, while those whichgive a negative pepton test are highly acid and the color produc-tion is rapid and profuse.

153


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


SUMMARY

One of the disadvantages of Endo agar as a differential mediumfor use with the colon-typhoid group of bacteria is the return ofthe fuchsin color in the agar of both inoculated and uninoculatedplates. This color may spread into the colony and hamperidentification.

These experiments were conducted to find out, if possible,what factor or combination of factors caused the return of colorand what could be done to prevent it. The formula recommendedby the Committee on American Standard Methods of WaterAnalysis (1923) was used for preparing the agar for these tests.Certain modifications recommended by different workers werealso tried, but they seemed to have no advantage over theStandard Method for eliminating color diffusion.The effect of different brands of pepton, of different kinds and

dilutions of basic fuchsin and of sodium sulphite and sodiumbisulphite as decolorizing agents were tested. The reactionof the agar and the effect of exposure to light were also considered.

It was found that the two most important factors in colorproduction seemed to be the kind of pepton and the strength ofthe alcoholic solution of the basic fuchsin, used as an indicator.Peptones giving a positive biuret test seemed to inhibit thecolor production more definitely, than those which gave a nega-tive test for pepton content.The American fuchsins differ in their solubility in alcohol so

that a fuchsin carefully tested for the proper dilution is less aptto cause return of color in the agar. Sodium sulphite is far moresatisfactory as a decolorizing agent than sodium bisulphite.The reaction of the medium is also important and should be

adjusted to at least pH 7.4 as acidity causes a rapid return ofthe color. Exposure to light is a fairly negligible factor as com-pared with the others.

CONCLUSIONS

This study of color production in Endo agar has not entirelysolved the problem, but it is clear that it offers certain possibilitieswhereby the condition may be improved.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


Endo agar is an excellent medium for detecting the presenceof and differentiating between different members of the colongroup of bacteria. In order to secure clear cut results, it isdesirable to have as little restoration of color in the agar aspossible, during the incubation period. Several factors seem toaffect this return of color, many of which may be modified orcontrolled.The peptones used in preparing the agar may have an effect

on the production of color and this seems to be in direct correla-tion with their actual pepton content.The reaction of the medium is important in checking color

diffusion. A decidedly alkalin reaction, ranging from pH 7.2to 7.6, does not affect the growth of the bacteria and, at thesame time, prevents a too rapid return of color in the plates.The strength of the fuchsin solution affects the production of

color. Since the different lots of American fuchsins, appearingon the market, differ in their solubility in alcohol, it is moresatisfactory to use a fuchsin which has been carefully tested forits ability to be decolorized in the sodium sulphite solution.The effect of light on the return of color during the first twenty-

four hours is fairly negligible, and, if other factors are carefullycontrolled, this factor need not be considered. By adding thefuchsin-sulphite solution immediately preceding the pouring ofthe plates any problem of return of color during storage may beehiminated.

Various modifications of Endo agar have been suggested, butnone of them seem to have any marked advantage over thestandard method. On the contrary, some of them do not appearto be as satisfactory as the standard method.Endo agar, made up with a high grade of pepton and a care-

fully tested solution of fuchsin, together with a carefully adjustedalkalinity, is an excellent differential medium for studying mem-bers of the colon-typhoid group of bacteria.

REFERENCES

CHAMOT, E. M., AND SHERWOOD, C. M. 1917 Culture media employed in thebacteriological examination of water. Jour. Amer. Chem. Soc., 39,1755-1766.

155


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


CONN, H. J. 1922a American biological stains compared with those of Grubler.Science, 55, 284-285.

CONN, H. J. 1922b Preliminary report on American biological stains. Science,56, 156-160.

CONN, H. J. 1923a The preparation of staining solutions. Science, 57, 15-16CONN, H. J. 1923b Dye solubility in relation to staining solutions. Science,

57,638-69.CONN, H. J. 1924 A report on basic fuchsin. Science, 60, 387-388.CONN, H. J. 1925 Biological Stains. The Commission, Geneva, N. Y.DEBoRD, G. G. 1917 The-fuchsin-aldehyde reaction on the Endo medium.

Jour. Bact; 2, 309-314.ENDO, S. 1904 Uber ein Verfahren zum Nachweis der Typhusbacillen. Cent.

fur. Bakt. I. abt., 35, 109.{GENUNG, E. F. 1924 Endo agar as affected by peptone. Science, 59, 282.GENUNG, E. F. 1926a Basic fuchsin as an indicator in Endo's medium. Stain

Tech., 1, 41.GENIUNG, E. F. 1926b A consideration of French's test of basic fuchsin for

Endo's medium. Stain Tech., 1, 135.GREY, E. C. 1913 The production of acetaldehyde during the fermentation of

B. coli communis. Biochem. Jour., 7, 359-363.HARDING, E. R., AND OsTENBERG, Z. 1914 Studies on Endo's medium with

observations on the differentiation of bacilli of the paratyphoid group.Jour. Infect. Dis., 11, 109.

HARRIS, N. MAcL. 1925 The preparation of Endo Medium. The MilitarySurgeon, 57, 280.

JORDAN, E. 0. 1924 General Bacteriology. Saunders Company.KAsTLE, J. H., AND ELVOVE, E. 1909 On the use of anhydrous sodium sulphite

in the preparation of Endo's medium. Jour. Infect. Dis., 69 619.LEVINE, MAX, 1921 Bacteria fermenting lactose and their significance. Iows

St. Col. Eng. Bul. 62.ROBINSON, H. C., AND RETTGER, L. F. 1916 Studies in the use of brilliant

green and a modified Endo's medium in the isolation of Bacillus typho-8US from feces. Jour. Med. Res., N. S., 29, 363.

Standard Methods of Water Analysis. 1923 Amer. Pub. Health Assoc., NewYork.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

Documents

AND DIFFUSION IN ENDO AGAR THEORIES FOR COLOR PHENOMENA IN ENDO AGAR Thereason for this color phenomenon has been explained in various ways by different workers, Endo (1904) explains