(FROM T H E BIOTECHNICAL-CHEMICAL 12,.1BOHAIORY, TECHNICAL COLLEGE OF D E N M A R l i j

ON THE COLIFORM BACTERIA OF HUMAN FECES By Erik Olsen.

(Received for publication November 19th, 1944.)

In connection with an extensive investigation of the flora of the intestine of elderly people, especially the so-called acidophilic, i. e., the lactic acid bacteria, that was carried out in collaboration with Professor Orla-Jensen, chief of the laboratory, and Dr. T. Geill, Physician-in-chief to the >>Old People's towncc in Copenhagen, to be published in Det Kgl. danske Videnskabernes Selskabs Skrifter, a quantitative as well as a qualitative investigation of the coliform bacteria in a great number of samples of feces w-as made. Of these specimens, 104 were more cIosely examined.

In the following a description is given of a n improved method for quantitative determination of Bact. coli and the like in feces as well as the applicability of this method in other fields. Further, in order to get an idea of the stability of the methods of identification, a survey is given of the coliform flora in the examined samples, the variation in the flora of 6 persons who were frequently examined during 3 months, and the results of repeated examinations of a number of strains after 1 and 2 years' cultivation in the laboratory.

For the determination of the number of bacteria belonging to the coli- aerogenes group, Durham tubes with lactose-peptone broth, as used in the examination of drinking-water according to the Danish standard methods, were employed at first.

As it was necessary to use 5 parallel tuhcs of each dilution to obtain a fairly accurate ))most probable<( number after McCrady's tables, it required rather a great deal of work and a large amount of culture medium to carry out this examination, the number of bacteria in the feces being so variable that one has to use a fairly large number of dilutions. Furthermore, in this way it is difficult to detect differenl species e\rntually present, on account of differences in their initial number, rate of growth, etc.

So it was tried to spread the material on a selective substrate, Levines eosin-methylene blue agar (in the following referred to as E-M agar) pre- parecl according to Danish standard methods.

In the beginning the usual metliorl of spreading was employed, 1 cc. of the diluted fecal suspension being pipettcd into the Petri dish and here mixed with the melted and cooled agar. Yet this appeared to give very

Acta path. Vol. XXII. 2 8

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poor results, partly because the numbers found were much too low and varying upon parallel plates, partly because the colonies were not charac- teristically developed, so that it was impossible with certainty to distinguish between deep colonies of Bact. coli and aerogencs.

To obtain more characteristic colonies it was therefore tried to smear 0.1 cc. diluted fecal suspension on the surface of E-M agar plates by means of a abutton spatula(( (Knopfspatel) ad modum Weissl.

Quite surprisingly, it then appeared that not only were the colonies beautifully developed and well separated but the number was usually con- siderably greater than the corresponding number found in Durham tubes, 5 tubes being used for each dilution; also there was a good conformity between parallel plates. The number was always greater on ))smeared(( than on the corresponding nspreada plates. The cause of this possibly is that more single cells are separated from chains and lumps by thc very thorough smearing that must be continued until the agar has absorbed all the liquid, than by the usual shaking in the dilution water.

The procedure is as follows: 10 cc. of agar are poured into Petri dishes after cooling to 45-50° C. to avoid too much water of condensation, and the surface of the agar is dried. The medium being very selective, this drying may without danger of infection take place with the lid somewhat tilted, for instance, a couple of hours a t 37O C. With less selective media this method cannot of course be used; they may either be dried with the lid tilted in a sterile Hansen box, by standing for a longer time with the lid on, or by replacing the glass lid with a porous one.

After desiccation the plate is inoculated, 0.1 cc. of the suspension being placed in the center of the plate. This is the most suitable amount of water for a dish with 9 cm. diameter; with a greater amount it takes all too long before it is absorbed, and a smaller one gives too large a n error in measur- ing. The remaining steps are exactly as described by Weiss, the flamed and cooled ))button spatula(( (consisting of a little flat glass button, the top of which is drawn out and bent to form a hook) by means of a rigid platinum or iron wire being placed in the droplet, which then is rubbed in by the weight of the spatula itself, with continually rotating movements of the spatula and simultaneous turning of the dish until all the liquid has been absorbed, which is easily felt when the spatula adheres to the agar. In order that this procedure shall not last too long, it is necessary that the surface of the agar be thoroughly dry.

During the smearing the lid of course has to be raised a considerable distance for about one minute, which does not involve any appreciable risk of infection with E-M agar, but with other media the work must be done in a Hansen box or similar contrivance.

The disadvantages of the method are that it takes a longer time than ordinary spreading and that only 0.1 cc. of the liquid can be used, an amount that is usually too small for the analysis of drinking-water, but for the analysis of sewage and water from purification plants with some hundred or thousands of cells of Bact. coli per cc. this method has proved fully equal to the dilution method in Durham tubes (usually not more than 1-2 dilu- tions are necessary on the plates, while more dilutions are always required in Durham tubes).

In the following some results characteristic of the numerous ex- periments made will be cited. Experiments with Fecal Suspensions (Table 1 ) :

The count in Durham tubes with lactose-peptone broth is carried out with 5 tubes of each dilution. The smears are made in duplicate

105

and both numbers are given. The recorded figures are the numbers of germs per g. of feces.

Table I Experiments with Fecal Suspension.

Sample 1 I Sample 2 1 Sample 3 1 Sample 4

Durham tubes 450,000 1,500,000 900,000 15,000,000 E-M agar, smeared 1,800,000 14,000,000 2,400,000 210,000,000

9 , ,9 1,100,000 13,800,000 2,100,000 184,000,000 ,, spread <100,000 800,000 2,100,000 50,000,000

Experiments with pure cultures of Bact. coli have given quite corresponding results, yet it should be mentioned that a single strain apparently particularly sensitive to methylene blue in several ex- periments always gave lower counts on E-M agar than in Durham tubes. Apparently the medium has not been suitable for it, smears on meat-peptone agar giving considerably higher counts than Durham tubes and common spreading on meat-peptone agar.

Otherwise, on comparison with other media as, for instance, meat- peptone agar, casein-peptone-yeast autolysate agar and lactate-glutamic acid agar after Eolpmers,2 E-M agar has given as high, and often higher, counts than these media.

A t 37O all colonies have grown out after about 40 hours’ incubation. According to my experience, the method is also suitable for the

isolation of pure cultures; on a second spreading of some 200 strains thus only one was found not to be pure. It contained enterococci.

In order to test the applicability of the methods in other fields, some orientating experiments with counts in milk have been made, spreading and smearing being compared. The counts have been madc in duplicate and the plates incubated at 30°. They were counted after 24, 48 and 72 hours’ incubation.

The figures given are the numbers of colonies on the plates (Tables 2 and 3).

Previously I have mentioned the selectivity of E-M agar and shall now go a little further into this question. With spreading from feces sometimes tiny, almost black, metal-shining colonies of enterococci, especially Sc. faecium, less frequently Sc. glycerinaceus and lique- faciens develop, besides the characteristic coli-aerogenes colonies. The streptococci of saliva and the pharynx, Sc. salivarius and pharyngis, may form the same sort of colony. Also various fluorescent bacteria, e. g., Ps. aeruginosa (Bact. pyocyaneum) may appear, but their large, irregular salmon-coloured colonies are easily recognizable.

a *

106

Dilution 10-3

Average

Dilution lO-4

Average

Table 2. Raw Whole Milk Spread and Smeared on Casein-peptone-yeast

Autolysate Agar.

24 hrs. 1 48 hrs. 1 72 hrs. 24 hrs. I 48 hrs. 1 72 hrs.

300 350 350 800 808 808 275 330 330 750 750 750

288 340 340 775 779 779

21 28 28 69 75 75 26 30 30 68 73 73

24 29 29 69 74 74

Smeared I Spread I

Dilution lO-3

Average

M-p agar C-y agar

Spread Smeared Spread Smeared 24 hrs. 1 48 hrs. 24 hrs, 1 48 hrs. 24 hrs. I 48 hrs. 24 hrs. I 48 hrs.

72 110 250 264 80 124 138 224 121 160 185 204 99 158 129 173

97 135 218 234 90 141 134 199 -___-------

As to the colonies of coli, aerogenes and intermediate types, an atypical appearance is rather often met with.

So transitional forms between coli and aerogenes colonies are rather often found. They usually behave as typical fecal coli bacteria. Further, quite white colonies of Bact. coli and aerogenes, besides blue and pink coli colonies are met with. In most instances the atypical type of colony has appeared on renewed spreading after 1 year’s storage in the laboratory, and by comparison with Endo agar, the atypical type has practically always asserted itself there too. 147 strains have been compared by spreading on EM and Endo agar; according to this material there are more atypical colonies on Endo than on E-M agar.

107

Quite peculiar behavior has been noticed in many of the coli strains that have at first formed aerogenes-like colonies, as they have grown strongly mucilaginous and viscous even in sugar-free broth. On Endo agar they are decidedly aerogenes-like, while on E-M agar they are more like coli bacteria. Physiologically these bacteria behave like typical fecal coli bacteria. On staining with crystal violet and counter- staining with nigrosin, distinct capsules are seen, giving the impres- sion that i t is a bacterium of the Friedlaender group (Klebsiel la) .

A similar behavior as to formation of mucilage has appeared in a number of strains which on examination immediately after isola- tion showed growth in Koser’s citrate medium, but on later examina- tion had lost this property completely. Even in protracted experiments to regain this property, starting with small amounts of lactose broth added to the citrate medium, only 1 of the 20 strains tried in this way regained the ability to grow in citrate. I therefore was inclined to doubt the correctness of these first examinations performed 3 years ago, but lately, from the lake in 0stre Anlaeg (Eastern Park) with a rich fauna of wild fowl, I have isolated a number of coli strains probably originating from the feces of the birds, which fully confirm the mentioned results.

On spreading on E-M agar the colonies were all alike but on comparison of 10 colonies it appeared that, as to the growth in citrate, 5 behaved as typical fecal coli bacteria, i . e. , citrate-negative, while the remaining 5 showed vigorous growth in citrate; in other respects they were like fecal coli bacteria. All media for identification were inoculated directly from the colonies on E-M agar and the purity was controlled at the same time by renewed spreading. To test the stability of the abilitx to grow in citrate, inoculations were made from the citrate media into fresh citrate and upon slanted meat-peptone agar, and from here again into citrate. In no case was growth obtained herein, so the power to utilize citrate is lost already in the second generation.

On going through the literature concerning the citrate test, it is seen that most authors have found it very stable. Thus Kosere has not observed any alteration in the behavior towards citrate by some 200 strains kept for 6 months - 2 years in the laboratory and re- inoculated every six weeks. Nor did keeping €or several months in water, earth or fecal suspension alter the state: only in 1 case did a strain of Aerogenes which originally did not grow in citrate, acquire the power to do so.

Parr & Caldwell” and d’Herelle & Rakieten5 have advanced the theory that there is a possibility of biochemical variation in freshly isolated strains that have not yet been accustomed to laboratory media. Stuart et c11.~, who, like Koser, find that the ability to grow in citrate is altered only in a few cases and then usually so that the strains which have not possessed this ability may acquire it, prefer to use

108

the expression >>stabilization(( instead of >)purification(( because they worked with pure cultures and >>purification< alludes to infection in the culture.

The described experiments with the citrate test seem to confirm the theory of these authors; only after stabilization of the culture may one reckon with correct results of the identification. So this ought not to be performed directly from the first spreading but the culture should be spread or reinoculated once or twice before identi- fication.

Yet it must be remembered that by far the greater part of thc abnormal strains mentioned here have developed a strong power of capsule formation after the rest of their properties were as in typical coli bacteria. Only a couple of the strains which have constantly behaved as typical coli have developed the power of capsule fornia- tion.

Smith & Orcutt’ and P a n 8 have found capsule-forming coli bacteria, Unfortunately, their papers have not been available so I know nothing of the history of these bacteria. and * are cited after P a d s very good symposium on the coli group!

So it seems very likely that here we have to do with transitional forms to the Friedlaender group (Klebsiella).

This group is somewhat varying in its physiological properties, yet on the whole it behaves as the coli-aerogenes group; capsule formation is a characteristic feature. According to Topley & WilsonlO (p. 526-534 and 571-572), it produces acid and gas by fermentation of various sugars, among these usually lactose, glucose, sucrose and salicin. It is indole-negative, methyl red-positive, Voges-Proskauer- negative, but usually citrate-positive, and so behaves as a coli-aerogenes intermediate (Escherichia freundii’ ) ) , apart from the capsule forma- tion which yet may disappear ( R type) or be regained (S type).

Both Parre and Topley & WilsonlO also find it difficult or even impossible to separate Klebsiella from the coli-aerogenes, group, especially Bact. aerogenes and intermediate types.

Thjottall is of the same opinion; he mentions, for instance, that the mucoid property may be induced to normal coli character by cultivation in a bacteriophage suspension or in an immune serum produced with the treated strain. Bacteriophages often occurring in inflamed localities, he does not find it improbable that a normal coli may change into a mucoid type. If it also alters its biochemical pro- perties, e . g . , by stopping indol-formation, not an unknown behavior,

’) The term Escherichia freundii as- a nomen for the intermedidte types has for the first time been used in the 5 edition of Bergey’s Manual of Determinative Bacteriology. Even when the genus names Cscherichia and Aerobacter otherwise have not been used in the present work, it would liardly be justifiable to make use of the term Duct. freundii, for which reason the American nomenclature is retained for this species.

it will after all be very difficult to separate such a bacterium from the so-called Klebsiella. So Thjotta considers Klebsiella indol-negative, while Topley & Wilson consider it variable. All species in Bergey’s manual are indol-negative.

I am not sufficiently acquainted with the medical bacteriology to be able to decide on the attitude to adopt towards the question, but I have found it of interest to mention the fact described here, namely, that initial citrate-positive strains develop into mucoid types by cultivation on common agar slants, and it seems to me that the observation supports the opinion that Klebsiella may hardly be con- sidered entitled to have its own genus as in Bergey’s system.

In the following n surrey is giren of the results obtained by ex- amination of the coliform flora of 104 samples of feces from 67 persons, mainly old people: 144 strains were examined.

The determination of the cultures was carried out by means of the methods usually employed; the criteria used were indol formation in trypsin-casein broth, methyl red test in Clarc & Lubs’ medium after 4 days at 37”, Voges-Proskauer’s reaction for acetyl-methyl- carbinol carried out in the following way: about 1 cc. of a 48-72-hour- old culture is mixed with 1 cc. 40 76 KOH in a white porcelain dish or a watch glass and some grains of creatin are added as given by O’Meara.12 In the course of 10--30 minutes a more distinct reaction is obtained than by the common method (mixing in a test-tube with equal parts 10 76 KOH and reading after 24 hours). Yet it is neces- sary to keep an eye on the process, the red color rather rapidly reaching a maximum and then decreasing.

With regard to the citrate test in Koser’s liquid medium, here, of course, it is of the greatest importance that no other carbon source be added, for instance, by inoculation from liquid media. I have in all cases inoculated from agar cultures suspended in sterile water.

These are the four, now almost classical methods, which ParrO with a good (or at any rate, practical) abbreviation has called irnuic quartet (indol, methyl red, Voges-Proskauer, citrate).

Further are included liquefaction of gelatin and motility, besides, for comparison with older methods, the fermentation of sucrose and salicin ad modum Winslow et al.lS, who set up the following types (Table 51, according to their opinion sufficiently stable to merit sepa- rate consideration.

It will be noted that the reactions in dulcitol and adonitol are included, but the species are adequately defined by the reactions in sucrose and salicin together with the motility.

In a recent paper by Kauffmann & Perch14 a long series of sugars has been employed with the result, that adonitol, dulcitol, rhamnose, salicin and saccharose together with indol and H,S formation gave almost as many types as serological reactions with antigens 0. Thus

110

Table 5.

Salicin Dulcitol Adonitol Motility S a c c h a - I rose I ! I Bact. neapolitanum

8 coli communius * coscoroba 8 coli commune

immobile acidi lactici

B grtinthal

in K 10 serological and 9 fermentative, and in P 22 serological and 20 fermentative types, were found while by application of saccharose, salicin and indol, only 4 and 8 types respectively would have been found. If motility also had been taken into consideration, there would, of course, have been a chance for a considerably greater number of types.

Nowadays not much stress is laid on motility, but as far as my experience goes, absence or presence of this property is very constant; in practically all cases it has remained unaltered after 2 years’ cul- tivation (see below). ’

The fermentation of cellobiose is an important criterium, B a d . aerogenes and Esch. freundii fermenting it, Bact. coli not. Unfortunate- ly, cellobiose has not been available, so this test had to be left out.

In the opinion of some authors, the formation of H,S is a means to differentiate between Esch. freundii and Bact. coli, yet this depends upon the method employed. In lead acetate agar there seems to be a difference, Esch. freundii forming H,S, but with lead acetate paper in the plug of the tube H,S formation is obtained with practically all coli and aerogenes species - as is also pointed out in Bergey’s manual, 5’ edition. So this reaction depends more upon a quantitative than a qualitative difference. Besides, it may be mentioned that the American manual of Methods for Pure Culture Study of Bacteria, 7’ edition, leaflet V, 1939, in contrast to earlier editions leaves out lead acetate agar and only recommends lead acetate paper.

According to the imvic methods, the different types of the coli- aerogenes group have the following properties 10 (P. 1578) (see Table 6 ) .

In the following survey of the material it is specified how the types found are distributed on the 104 samples of feces examined (Tables 7 and 8 ) .

*) A = acid, G = gas, - = no fermexhtion.

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Table 6 .

Indol Methyl Citrate Gelatin I ~ red 1 V’-p’ 1 1 liquefac.

- - - Bact. coli, fecal type I ...... + + +

+ Intermediate, type I - + + freundii )) I1 + +

...... + + Bact. aerogenes, type I - -

)) )) )) I1 ...... + + + .................. + + + Bact. cloacae -

- - - -. )) )) )) )) I1 ...... - - - - - - -

Table 7. Imvic Types.

Number % 59.7

)) )) )) >) )) )) I , and citrate-pos. later citrate-neg. .. 22 21.2

)) )) )) )) )) )) I, and fecal type I1 . . 4 3.8 )) )) )) )) )) )) I , and Esch. freundii . 2 1.9 )) )) )) )) )) )) I, and Bact. aerogenes

)) >) )) )) )) )) I, and Bact. cloacae

)) )) )) )) )) )) I, here included

Samples with Bact. coli, fecal type I , alone .............. 62

(2-80 % aerogenes) 10 9.6

(14-50 % cloacae). . 2 1.9

citrate-pos.which . . later became citrate- neg. ................ 102 98.1

)) )) Bact. aerogenes alone ...................... 2 1.9 104 100.0

~ ~~

>>Slow lactose fermenterscc were only found in 2 samples = 1.9 %.

Table 8. Types after Winslow ct al.

Bact. neapolitanum ...................................... )) coli communius .................................... )) coscoroba ........................................... )) coli commune ...................................... )) immobile ............................................ )) acidi lactici ........................................ )) griinthal .............................................

Divergent type (motile, saccharose and salicin-pos.) ......

Number % 11 9.8 9 8.0 7 6.3

19 17.0 20 17.9 22 19.6 10 8.9 14 12.5

112 100.0

Types after Winslow ef al. (compare Table 5 ) . (Table 8 . ) I n some cases several different types have been found in the same

112

sample of feces, resulting in a larger number of strains than of samples, even when the aerogenes and cloacae are left out. In order to get an idea of the frequency of the different types, the percental calculation has therefore been made on the enumerated 112 cultures (where more strains of the same type have been isolated from one sample they have, of course, only been included once).

It will be noticed that commune, immobile and acidi lactici are the most frequent types.

Bardsleyls, examining 47 samples of feces, found Bact. coli in 94 %,

Intermediate (imvic --f--+) in 16 % )) aerogenes in 9 %, and

by direct spreading on McConkey agar.

after 8 hours’ enrichment in lactose broth, found: Carpenter & Fultonl* have, by spreading 466 samples on E-M agar,

Citrate-positive (aerobacters and intermediates) in 49.8 % Bact. coli alone in 47.6 7%

The intermediates were usually always found in the same persons.

the following (Table 9 ) : Pan1’ has smeared 235 samples of feces on Endo agar and found

Table 9. No Bact. coli-aerogenes in .................. 6.8 $6 Only coli in .................................. 55 3 % Coli and aerogcnes in ........................ 17.4% Coli and intermediates in .................... 10.6% Coli and intermediates and aerogenes in .... 8.0 % Only intermediates in ........................ 0.4% Only aerogenes in ............................ 0.4 % Intermediates and aerogenes in .............. 2.1 % Cloacae in .................................... 0.4 % Slow-lactose coli in .......................... 5.9%

So, apart from the very large number of citrate-positive strains found by Carpenter & Fulton, the results obtained in the four cases are all of the same magnitude.

In a general way, then, it seems that conclusions can be drawn to the effect that the fecal type of B a d . coli occurs in nearly all samples of feces, but in about one-half of the samples it is found together with types that are not usually regarded as being of fecal origin - Bact. aerogenes, intermediate (Esch. freundii) and Bact. cloacae. The amount of these non-fecal types varies from 2 to 80 % of the total coli flora. A pure aerogenes or intermediate flora is comparatively rare but the occurrence of such cases shows that i t can never with absolute certainty be decided, for instance, whether or not a sample of water is polluted fecally, even i f fecal coli bacteria are not found.

113

In order to ascertain how coilstant or variable is the coli flora in the same person during a longer space of time, samples of feces from 6 persons were examined every 1-2 weeks during about 3 months, 2-4 colonies being picked from each sample depending on the uniformity of the colonies. Rather great variations were thus found in the composition of the coli flora from time to time when the determination was made after the system of Winslow et al. But after the imvic system no essential variation could be found; in almost all cases fecal type I of coli was found and in person 2 Bact. aerogenes was found regularly in an amount varying from 1 to 50 % of the total coli flora on E-BI agar.

In Table 10 the imvic type is recorded and in parenthesis the Winslow type, partly giving the numbers of the different types where this has been possible by means of the different appearance of the colonies. The strains marked with X are of the diverging type already mentioned a t the bottom of Table 8 ; it ferments both sucrose and salicin, but, in contrast to Bact. neapolitanum, it is motile. Moreover, this type seems to be rather constant, only one of the 12 strains mentioned here having changed its properties (into Bact. coli com- mune) after 2 years’ cultivation. All strains marked as citrate-positive have on later examinations lost this property. Only in one case (per- son 3, 1st. time) was this property retained, the culture therefore being listed as Esch. freundii.

From Table 10 it is seen that the flora usually differs from time to time, and often more types are found at the same time. Yet a great uniformity is found in the flora of person 2, Bact. neapolitanum and aerogenes being found constantly.

Similar results have been obtained by Parr17 and by Kauffmann & PerchI4 who, as already mentioned, found numerous types in 2 persons; several types were present at the same time and they varied from time to time.

In identification of bacteria, of course, it is of great importance that the characteristics employed are fairly constant. The bacteria of the coli group are very inconstant in many respects, yet most of the imvic tests seem to be rather constant. Most work has been done on the correlation between the different tests, but as to the citrate test Koser3 and Stuart e f al.O as previously mentioned have found a great constancy; if alterations do occur, it is mainly in a positive direction, i. e., strains that have not been able to grow on citrate may acquire this ability.

Nyberg et a1.18 examined 138 strains of the coli-typhoid-dysentery group isolated from sewage and cultivated for 2 years in agar stabs in the laboratory. On renewed spreading several cultures split up into more variants, one of which was often, but not always, like the original culture. Other cultures changed completely. Altogether

114

Table 10.

Fecal I 'communius

and X)

Fecal I (commun-

ius)

Fecal I (commun-

ius + 17Oic acidi lactici

- __

- l'tim

- 2'tim

- 3'tim

- 4'tim

- 5'tim

7

'erson 1 Person 2 Person 3 Person 4 Person 6 Person 5

Fecal I (commun- ius) + 30 010

freundii :coscoroba)

Fecal I commun- us and X) itrate-pos.

(XI

Citrate- positive

:oscoroba). Imvic ++--

;elatin liq. (XI

Fecal I immobile)

Fecal I (XI

-

Fecal I (neapoli- tanum)

Citrate- positive

:coscoroba:

Fecal I (neapoli- tanum) + 50 Oio .

aerogenes

Fecal I (neapoli- tanum) + 2010

aerogenes

Fecal I (X)

Citrate- positive (neapoli- tanum) + 1010

aerogenes

Citrate- positive (neapoli- tanum) + 20 o/o

aerogenes

Fecal I (neapoli- tanum) 4- 40 Oio

aerogenes

Fecal I neapoli- tanum)

Fecal I :coxor-

oba, ommun. ius, X)

40% fecal

citrate-pos. (neapoli- tanum)

I (X) 6OOio

Fecal I (coscor-

oba) + 50 O i o cloacae

Fecal I (immobile)

Citrate- positive

(immobile:

Fecal I (neapoli- tanum)

Fecal I1 (acidi

lactici)

~~

Fecal I (commune 4- 50 O I o X)

Fecal I (X)

Fecal I (X)

Fecal I (acidi

lactici)

Fecal I (acidi lactici)

Fecal I (neapoli- tanum) + 4% imvic -++-

Fecal I (com- mune)

-

-

Fecal I (commun-

ius) + 15 Oio citrate-pos. (neapoli- tanurn)

6'tim

- 7'tim

-

Fecal I (neapoli- tanum) + 14 O1o

aerogenes

750/0 citrate POS. (X)

25% fecal (neapoli- tanum)

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there was a great capacity for changing in this very heterogenous material, which the authors partly ascribe to the fact that the bac- tecia in sewage have been in contact with bacteriophages. Most con- stant was the Voges-Proskauer reaction ( 5 changed), then methyl red (9 changed) and indol (11 changed); citrate was not tried. The fermentation of sugars was much altered, so 23 strains had changed against lactose, 14 against sucrose and 9 against dextrose.

A better basis for comparison with the cultures examined in the present work is Siever’si9 study on 7-year-old cultures of coli and kindred bacteria from human feces, urine, blood, etc.. 87 strains were alive, cultivated on agar slants. These strains had all been motile but on reexamination 42 had become immotile; otherwise only few changes had taken place, A s to the indol, M-R and V.-P. tests, 1 strain had changed in each, against lactose 4, salicin 7, sucrose 6, dextrose 0. Citrate was not tested.

So motility here has appeared very labile but as generally known this may be rather difficult to establish with certainty; the results most in accordance are certainly obtained when the same person makes the examination each time, which was not the case in Sever’s work. Curious, too, is the change in the ability to ferment lactose in 4 strains, 2 of which had lost and 2 acquired it.

In the present study experiments have been made with cultivation of the cultures on agar slants for 2 years and reinoculation every 6 months, and they were tested after 1 and 2 years’ cultivation. Un- fortunately, many of the cultures died between the first and second examination on account of desiccation. After 1 and 2 years’ keeping, 103 and 68 cultures, respectively, were tested according to the imvic quartet and for fermentation of lactose, sucrose and salicin; further- more, motility was studied.

In the following survey the number of cultures having changed the property in question is specified for each test (indol, M-R, etc.) as well as how they were classified at the first and second identifica- tions.

For all the imvic tests it may be said that when alteration has occurred after 1 year, i t kept unaltered till the next year. On the other hand, the fermentation of sucrose and salicin may change again.

Zndol: Of 12 indol-neg. 2 (fecal type 11) became indol-pos. (fecal type I ) . Of 91 indol-pos. 1 (aerogenes 11) became indol-neg. (nerogenes I ) .

Of 97 M-R-pos. 3 (imvic -+ + +) became bt-R-neg. (acrogeiics I ) . Of 6 M-R-neg. 1 (imvic --+-) became M-R-pos. ( ? ).

Of 95 V.-P.-neg. 1 (fecal type 11) became V.-P.-pos. (aerogenes I). 8 V.-P.-positive (6 aerogenes, 1 cloacae and 2 atypical (imvic -+--., after

M e t h y l r ed .

Voges-Proskauer:

1 year -++-) werc unaltered.

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Citra te: Of 27 citrate-pos. (Esch. freundii) 25 became citrate-neg. (fecal type I). Of these 2 were from the same sample of feces, and 1 did not change until

6 aerogones, 10 cloacae and 1 atypical (see Voges-Proskauer) were un-

69 citrate-negative were all unchanged.

Of 47 motile 1 (fecal type I , communius) became imniotile Ineapolitanum). 56 immotile were unchanged.

Sucrose: Of 46 sucrose-pos. 19 became sucrose-neg. after 1 year. 4 of these became

sucrose-pos. again after 2 years. From 1 person 7 cultures from different samples of feces were examined. They were all sucrose-pos. at first and sucrose-neg. after 1 and 2 years.

Salicin:

the second year.

changed.

Moti l i ty :

Of 57 sucrose-neg. 6 became sucrose-pos., 4 of them only after 2 years.

Of 59 salicin-pos. 4 became salicin-neg., 1 pos. again after 2 years. Of 44 salicin-neg. 9 became salicin-pos., 1 neg. again after 2 years. 1 became pos. only after 2 years.

So it seems that the stabilization of the cultures applies to all the imvic tests. This is especially true of the citrate test (d. p. 5 ) but also in a few cases to the other tests, particularly in less fre- quently occurring or entirely divergent types.

According to these results, the most constant reactions seem to be indol-pos., methyl red (it is true that 3 M-R-pos. strains have become M-R-neg. but they have all been isolated from different samples of feces from the same person, so here it seems to be a matter of a special type rather constantly found in this person). The Voges- Proskauer reaction has appeared very constant, whereas citrate-pos. in connection with indol-pos. and M-R-pos. has great chances of be- coming negative.

Motility is apparently a very constant property, 1 strain only of 47 motile having lost this property, while the immotile were all unchanged.

On the other hand, the fermentation of sucrose and salicin appears rather unstable, especially sucrose-pos. is often changed to negative and may again change to positive. 4 of the 19 sucrose-pos. strains that became negative originate from different specimens of feces from the same person, so it is possibly a type predominating in this person, only 1 culture from him having stayed positive all the time.

Yet the fermentation of lactose and glucose which have always been included in the examinations has appeared quite constant.

Such a great instability in the fermentation of sugars shows the justice of abandoning the different systems based on the fermenta- tion of sugars: but as shown by Wohlfeil & YaiIeP in the typhoid- paratyphoid-enteritis group it may depend on deficiency in cozymase while the apoenzyme ( j-h-fructosidase or a-glycosidase) is present.

By addition of a yeast extract containing cozymase, a vigorous

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fermentation may in such cases be obtained. So possibly it is small differences in the medium that determine whether the bacterium itself is able to form cozymase.

Motility seems to be so constant a property that it may well be given taxonomic importance.

Unfortunately, Eijkman’s test for ,warin-blood colic(, i. e., fermen- tation of glucose at 43-45O (Eijkman originally proposed 46n,, but this temperature is too high) was not carried through at the first examination, but one year later 25 of the cultures were examined for growth at 40-42-44-46O after inoculation in peptone water pre- heated to the respective temperatures.

After 72 hours’ incubation the following results were obtained:

Table 41.

Species

.................... Bact. coli, fecal type I 16 16 14 11 0 Citra.te-pos., later fecal type I . . . . . . . . . . . . . . 4 4 . 3 1 0 Bact. coli, fecal type I1 .................... 1 1 1 1 0 Esch. freundii .............................. 1 1 1 1 0 Bact. aerogenes ............................ 2 2 1 0 0 Bact. cloacae .............................. 1 1 1 1 0

This material is of course too small to allow of definite conclusions, but as it may be assumed that contingent changes in temperature maximum occur more rapidly in water than by cultivation at 37O, i t seems that some reserve is appropriate in judging of the Eijkman test where not very recent fecal pollutions of the examined water are involved, since, as is seen, 8 out of 20 B a d . coli (including the originally citrate-pos. strains) do not grow at 44O and 3 not even at 42O. At any rate, in all cases 46O is too high.

Summary. 1. It is shown that smearing on the surface of agar gives a greater

number of colonies than common spreading and the dilution method in Durham tubes.

2. Freshly isolated cultures have to be ))stabilized< by reinocula- tion a couple of times before identification, since misleading results otherwise may be obtained, especially in the case of the citrate test.

3. Many strains originally being citrate-pos. (or forming aerogenes- like colonies) have in the course of time acquired a power of capsule formation. The relation of these strains to the Friedlaender group

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(Klebsiella) and the right to put up Klebsiella as a special genus is discussed.

4. Bact. coli, fecal type I , has been found in 98 per cent of 104 samples of feces, in mixture with Bact. aerogenes in 9.6 per cent, and Bact. aerogenes alone in 2 per cent of the samples.

Of the Winslow types Bact. coli commune, Bact. immobile and acid!' lacfici were found most frequently.

5. In 6 persons examined several times variation of the types was usually found from time to time; as a rule, several types were present at the same time.

6. The stability of the reactions made use of has been tested by examination of the cultures after 1 and 2 years' cultivation.

The imvic tests are generally rather constant, especially the Voges- Proskauer reaction. The citrate test is reliable only after stabilization of the cultures.

The fermentation of lactose and glucose is constant while the fermentation of sucrose and salicin show great variation.

7. Eijkman's test for )>warniblood-colicc has been tried on l-year- old cultures. 8 out of 20 Bact. coli did not grow at 44O nor 3 at 42".

REFERENCES 1. Weiss, R.: Zentralbl. f. Bakt. I, Org. 442: 105, 1938. 2. Folpmers, T.: Antonie van Leeuwenhoek, 6: 22, 1940. 3. Koser, S . A.: J. Infect. Dis. 35: 315, 1924. 4. Purr, L. W. & Caldwell, E . L.: J. Infect. Dis. 53: 24, 1933. 5. d'Herelle, F . & Rakieten, T . L.: J. Infect. Dis. 54: 313, 1934. 6. Stuart, C. A., Griff in, A. M . & Baker, M. E.: J. Bact. 36: 391, 1938. 7. Smith, T. & Orcutt, M . L.: J. Exp. Med. 41: 89, 1925. 8. Paw, L. W.: Proc. SOC. Exp. Biol. Med. 31: 226, 1933-34. 9. - Bact. Rev. 3: 11, 1939.

10. Topley, W . W . C. & Wilson, G. S.: The Principles of Bact. and Inim.

11. Thjetta, Th.: Lserebok i Bakteriologi. Vol. 11, Oslo 1942, p. 259. 12. O'Meara: Brit. J. Exp. Path. .12: 346, 1931, 13. Winslow, W-E , A., Kligler, I . J . & Rothberg, W.: J . Bact. 4: 429, 1919. 14. Kauffmann, F . & Perch, B.: Acta path. et microbiol. Scandinas. 20: 801,

15. Bardsley, I). A.: J. Hyg. 34: 38, 1934. 16. Carpenter, Ph. L. & Fulton, McDonald: Am. J. Publ. Health. 27: 822, 1937. 17. Pam, L. W.: Am. J. Hyg. 27: 67, 1938. 18. Nyberg, C., Bonsdorff, K . & Kauppi, K.: Zentralbl. f. Bakt. I. Org. !39:

19, Sievers, 0.: ibidem, p. 27. 20. Wohlfeil, T . & YalZez, J. P.: Zentralbl. f. Bakt. I. Org. 454: 237, 1944.

2' edit. London, 1936.

1943.

13, 1937.