The interest of this case is due to the fact that the patient from whom this bacillus was isolated was a tabetic in whom a spontaneous fracture of his femur had occurred, and cellulitis developed at the level of the lesion due to a diphtheria-like bacillus. This organism was isolated and obtained in pure culture from the cellulitis and was found present in the films of the pus; also the patient's serum gave a complement fixation reaction with his own bacillus and for syphilis.

In this report are included a series of a thousand cases examined from Jan. 1910 to Jan. 1911, the tests being made weekly at the R. N. Hospital, Haslar. Most of the cases were under observation in the hospital when their clinical characters could be recognised; in many of the cases several tests were applied, week after week, thus making the results obtained of greater value.

1. A spontaneous epidemic of Gaertner infection among rats was found associated with methaemoglobinaemia and, in some cases, anaemia.

2. Strains of Gaertner's bacillus isolated from these rats reproduced methaemoglobinaemia in fresh rats but not in rabbits, guinea-pigs or mice.

3. Other strains of Gaertner's bacillus from rats, guinea-pigs and human sources also caused methaemoglobinaemia either before or after passage through rats.

4. Other organisms pathogenic for rats did not produce methaemoglobinaemia.

The present paper contains the results of an enquiry into the differentiation of certain organisms of varying source, which present characters more or less closely allied to those of the “mannite” or “Flexner” type of the dysentery bacillus.

The practice of flour bleaching originated in this country about ten years ago; bleaching, however, is said to have been attempted in France two or three years prior to this. The practice was rapidly adopted by millers in the United Kingdom, in America, and elsewhere. Such bleaching is applied only to roller-milled flour. Numerous patents have been granted for bleaching appliances and for the application of various chemicals to the bleaching of flour. Ozone, chlorine, oxides of chlorine, bromine, nitrogen peroxide and other oxidising substances have been suggested and tried as bleaching agents. Of these substances nitrogen peroxide is the only one which has given satisfactory results, and all commercial bleaching of flour at the present time depends on the use of this substance. It can be produced in various ways and from a variety of materials. Two methods of production, which may convniently be termed the chemical and electrical methods, are in use commercially at the present time. In the first of these nitrogen peroxide is produced by the action of nitric acid on ferrous sulphate, the amount of gas formed being controlled by regulating the supply of acid and ferrous sulphate. A current of air charged with the gas is led through a revolving reel or “agitator” as it is called, through which a stream of flour is continually flowing. The flour in its passage is throughly exposed to the action of the nitrogen peroxide, and is bleached when it emerges from the agitator. In actual practice this process possesses certain commercial disadvantages. The apparatus requires constant attention if the degree of bleaching is to be properly controlled; overbleaching and condensation of acid resulting in local staining of the flour are liable to occur, while in the hands of ordinary workmen the use of strong acids is not entirely free from risk. As regards expense, it compares unfavourably with other methods, some of which are said to be worked at one-tenth of the cost of the acid process.

The foregoing experiments conclusively show that a broth medium is quite unsuitable as a test culture medium to determine the vitality of anthrax spores in disinfection experiments, whereas agar is a suitable and delicate medium for the purpose, even when considerable traces of the disinfectant are carried over with the inoculation.

The reason for this inefficiency of broth is not obvious. We thought that it might be due to the absence of bacillar forms in the sporing material, but the emulsion of spores heated to 80°C. for 15 minutes and then inoculated directly into broth gave good growths.

Absence of oxygen might be another factor, but the results were the same when splinters of sterilised wood infected with anthrax spores were treated. The wood floated on the surface of the broth and so was subjected to a free supply of oxygen, yet no growths were obtained in broth when the splinters were soaked in the disinfectants, while good growths were obtained on agar. The control splinters gave good growths in broth. It may be that the anthrax spores are partially de-vitalised by the action of the disinfectant and that in this condition broth is a comparatively unsuitable culture medium for them. Prolonging the time of incubation of the broth cultures up to 10 or 14 days makes no difference. If a culture in broth shows no growth in 48 hours, a growth hardly ever appears with more prolonged incubation. Nor is this superiority of agar over broth as a culture medium confined to the emulsified disinfectants employed in these experiments, for similar results have been obtained with phenol, and with formaldehyde, the latter both in the fluid (formalin), and in the gaseous, conditions.

Although typhoid bacilli were liberally supplied to larvae of Musca domestica, all attempts to demonstrate B. typhosus in the pupae or imagines were unsuccessful, until recourse was had to disinfection of the ova. After this preliminary disinfection both larvae and pupae gave pure growths of B. typhosus but hitherto it has not been possible to examine the imagines.

In the experiments with unsterilised ova great difficulty was experienced in determining whether B. typhosus was present in MacConkey plates owing to the almost invariable occurrence of the colourless typhoid-like colonies of the Bacillus “A” which was evidently an organism throughly adapted to the conditions prevailing in the interior of the larvae, pupae and imagines.

The fact also that this organism like B. typhosus fermented mannite without the production of gas, rendered the search for B. typhosus still more difficult.

By the employment of sorbite—in place of lactose-media, a preliminary differentiation might have been possible but was not considered practicable in the first place owing to the expense of sorbite, and secondly owing to the fact that the fermentation of sorbite by B. typhosus though constant, is sometimes delayed. The most satisfactory plan was to pick off as many colonies as possible and inoculate them on litmus-milk. Bacillus “A” invariably rendered litmus-milk intensely alkaline.

From the practical point of view the main conclusion to be drawn from the experiments detailed in this communication is that the typhoid bacillus can lead only a very precarious existence in the interior of larvae or pupae which possess, at least in so far as these investigations warrant, a well-defined bacterial flora of their own.

Even under the highly artificial conditions of the final series of experiments, it was not possible to decide whether the B. typhosus though recoverable from the pupa was really actively multiplying in the pupal interior or gradually dying out. There was some indication that the latter was the case, as the typhoid colonies recovered from the pupa in the one successful instance, were extremely few in number, while the larvae which had been feeding on B. typhosus contained enormous numbers as evidenced both by cultural and microscopical examination.

Since the above experiments were concluded, some further researches by Graham-Smith (1911) on the carriage of bacteria by flies have appeared in the form of a Local Government Board Report. This author has succeeded in recovering B. anthracis from blow-flies bred from larvae fed on meat infected with spores of this organism, but has failed to recover B. typhosus or B. enteritidis under similar experimental conditions.

On June 14th, 1910, the attention of one of us was directed by the House Physician of the Hampstead General Hospital, to an outbreak in Paddington of what was thought to be “food-poisoning.” Two of the patients were at the time under his care.

(1) B. typhosus ferments dulcite-peptone-water in about 10 days. By sub-culture this is readily shortenedto one day. It produces nonfermenting colonies on dulcite-agar plates. The colonies show after five days and upwards, secondary colonies as papillae, which may or may not have an acid reaction.

Growth of B. typhosus on dulcite media readily gives rise to bacilli forming, from the commencement, acid colonies on dulcite plates. The dulcite character has a tendency to atavism in the early stage which further growth on dulcite removes.

(2) B. typhosus can be trained to ferment lactose, only with very great difficulty. Definite and conclusive results have been obtained by meonly with the white atavists of Twort's trained lactose strain. Other strains (20) have not yielded to such training methods, although many of them have been trained for over a year. The lactose character when acquired is very unstable, and is soon lost when growth takes place in a lactose-free medium. A fermenting colony on replating gives rise to a mixture of fermenting and non-fermenting colonies.

(3) All strains of B. typhosus which I have examined show the secondary colonies on isodulcite described by Reiner Müller. Growth on isodulcite yields directly a strain without this papillae-forming power. So-called “acid” papillae as described by Reiner Müller have not been observed in the strains (20) investigated by me. This new strain does not regain the power to form isodulcite papillae by frequent sub-culture apart from isodulcite. Apparently the acquired character has arisen by mutation. Fermentation of isodulcite requires a considerably longer process of training.

(4) The above three characters vary in permanency. The permanency varies inversely as the time required for the selection or training.

(5) Papillae-formation arising on carbohydrate media on colonies of members of the typhoid-coli group may indicate variation by slow degrees just as well as by mutation. I have observed it in normal typhoid varying towards dulcite and lactose as well as isodulcite. In the case of Twort's strain which fermented sorbite-agar.

Formation of papillae simply indicates apparently that the variation only affects very few individuals of the colony to a great extent, and does not guarantee permanency in the character.

If the papillae arise early and without sub-culture, the evidence adduced so far suggests that the character will have considerble permanency. In the case of dulcite the results show that the formation of papillae on certain carbohydrate media cannot, as Reiner Müller stated, be regarded as definitely specific for the variety of microorganism exhibiting them.

The variations detailed in the above communication do not in our view invalidate the sugar tests as aids to differentiation, since these chemical functions are subject to variation probably to no greater extent than other functions of bacteria. Further, the ability of an organism to vary in any particular direction may be of considerable value in differential diagnosis.

This instability makes it imperative to have as large a number of differentiating characters as possible for every species, and also to take care in laboratory culture that we do not grow bacteria under conditions likely to alter their characters. It would be inadvisable for example to propagate the B. typhosus from a dulcite culture a few days old if one desired it to conform to the normal typhoid type in respect of dulcite.

In sending this paper to the Press I would like to acknowledge my indebtedness to Dr Martin, who has supervised my work on behalf of the British Medical Association, and to Dr Ledingham, who suggested the work, from both of whom I have received much valuable help and criticism. Many other colleagues at the Lister Institute have helped me from time to time in points of difficulty, and I take this opportunity of thanking them.

The action of air containing nitrogen peroxide upon flour, in quantities up to 300 c.c. of nitrogen peroxide to one kilogramme of flour, may be summarised as follows:—

I. The golden-yellow tint of the flour is destroyed. Immediately after bleaching no difference in tint due to excess of the bleaching agent could be observed with Lovibond's tintometer, but on keeping for several days the more highly bleached samples became decidedly yellow, while those treated with 30 to 100 c.c. of nitrogen peroxide per kilogramme became still whiter, the maximum of bleaching effect being attained within these limits.

II. The amount of nitrous acid or nitrites present in a freshly bleached flour is approximately proportional to the amount of nitrogen peroxide employed, and corresponds to about 30% of the total nitrogen absorbed, rising to 40% in the more highly bleached samples. After the lapse of several days, the proportion of nitrites present decreases considerably in the higher concentrations, but remains very nearly the same in the more slightly bleached samples.

III. Approximately 60% of the total nitrogen introduced as nitrogen peroxide into the flour during bleaching can be recovered as ammonia a short time after bleaching by reducing the aqueous extract of the flour with a copper-zinc couple, and may be assumed to be present in the flour as nitric and nitrous acids or as nitrates and nitrites. After keeping the bleached flour for some days the amount of nitric acid extracted with cold water decreases. Experiments with pure glutenin and gliadin indicated that in certain circumstances nitric acid may be withdrawn from solution or “adsorbed” by these proteins.

IV. In highly bleached flour a considerable increase in the amounts of soluble proteins and soluble carbohydrates takes place. If one kilogramme of flour is bleached with 300 c.c. of nitrogen peroxide, the amount of soluble nitrogen is doubled. This appears to be due almost entirely to the solubility of gliadin in nitric acid of certain concentrations. The simultaneous increase of soluble carbohydrates would seem to point to an intimate relationship between the gliadin and certain carbohydrates in flour.

V. If highly bleached flour is allowed to stand for some time after bleaching, the oil undergoes very considerable alteration and acquires the characteristics of an oxidised oil. About 6 to 7% of the nitrogen introduced as nitrogen peroxide during bleaching is absorbed by the oil.

VI. The absorption of nitrogen peroxide by flour does not appear to be accompanied by the production of free nitrogen, nor was any evidence obtained of the formation of diazo-compounds.

VII. Sodium nitrite was found to exert no inhibitory action on the digestion of soluble starch by saliva, but the rate of digestion was greatly retarded if the starch had been previously treated with nitrogen peroxide gas. Bleaching was found to exercise an inhibitory effect on the salivary digestion of flour.

The digestion of solidified blood-serum by certain sporing bacilli is well known, and Bienstock (1899) and Rettger have shown that B. putrificus and other obligate anaerobic sporing bacteria destroy protein and cause putrefactive changes. Hardly any observations have been made, however, to determine whether aerobic, non-sporing bacilli and cocci have any power to break down proteid, although it is often assumed they can do so. Rettger (1906) found that bacilli of the colon group did not set up putrefactive changes in a mixture of egg and meat protein; he made no quantitative experiments as to whether any destruction of protein took place, and Dr C. J. Martin informed me that he has for long noticed that preparations of pure proteins, when left exposed to air, did not appear to be broken down by bacteria, although moulds were able to grow in them.

Infusoria have the same bactericidal power as flagellates. Emulsions containing Bacillus typhosus, Vibrio cholerae, V. Dunbar, V. El Tor, B. megatherium and Spirillum volutans, to which Colpoda cucullus is added, are soon cleared. Before the clearage the Colpodae multiply actively.

This bactericidal effect does not depend upon the production of toxic substances by the Infusoria. The fluid obtained from filtered cultures of the Infusoria exerts no bactericidal effect. Only living Colpodae are able to clear the emulsions.

Direct sunlight does not prevent the clearing of the emulsions by the Infusoria, but temperatures below 10°C. and above 30°C. and absence of oxygen (anaerobic culture) are unfavourable to their exerting a bactericidal effect.

The polluted water of the canals of Amsterdam slightly delayed the clearage of the emulsions; the sewage of starch factories and gas factories completely prevented this clearage.

Consequently under natural conditions Infusoria will only play a part in purifying river water (1) if the temperature is above 10° and under 30°C., (2) if the aquatic vegetation is rich enough to supply the necessary quantity of oxygen, (3) if the water is not highly polluted by adjoining factories.

In a recent communication (Penfold 1911) I described experiments with three organisms, B. coli Escherich, B. enteritidis Gaertner and B. Grünthal, showing that limitation or complete abolition of certain gas-forming powers normally possessed by these organisms, could be brought about by their growth on chloracetic acid-agar, and further, that in the case of the latter organism, resistance to this medium was associated with such limitation or abolition.

The results obtained in these investigations were negative throughout, but perhaps are worth recording as tending to show that the formation of such ‘anti-bodies’ as cytolysins and precipitins is not a necessary reaction on the part of any living organism to the presence of foreign cells or substances.

1. The term “paratyphoid group of bacilli” has been confined in the present enquiry to those strains which in their cultural characters and agglutination reactions are indistinguishable from B. suipestifer and B. paratyphosus (B). B. enteritidis Gaertner and B. paratyphosus (A) are excluded.

2. The strains of bacilli belonging to the “paratyphoid” group can be separated into two classes by means of the absorption method, namely one group identical with recognised, standard strains of B. suipestifer and one identical with standard strains of B. paratyphosus (B); and we regard these two bacilli as separate organisms.

3. In our experience, B. suipestifer has been found only in food or in outbreaks of acute illness attributable to food-poisoning, whereas B. paratyphosus (B) has been found in cases of paratyphoid fever or in persistent paratyphoid “carriers.”.

4. We are disposed to put forward the suggestion indicated by these observations, that these two organisms have a different distribution in nature, the normal habitat of B. suipestifer being the alimentary canal of the pig (and other animals) and of food derived from such animals, whereas the normal habitat of B. paratyphosus (B) is the human alimentary tract (including the gall-bladder).

5. The examination of cultures from the faeces and urine of 300 typhoid convalescents was completely negative, neither B. suipestifer nor B. paratyphosus (B) being obtained. These observations confirm those of other writers, and, apart from carrier cases, the occurrence of B. paratyphosus (B) in healthy human beings appears to be unknown in this country.

Our thanks are due to Drs Fisher, Trautmann, Uhlenhuth, Williams, Rommeler, Prigge, Schern, Friedrichs, Savage and Fowler, who have very kindly supplied us with strains of bacilli for investigation.

On February 10th, 1911, the attention of the Medical Officer of Health was called by the practitioner in charge to an outbreak of diarrhoea in a family named R. residing in Paddington. The note made at the interview was to the effect that cases had occurred on the 8th, 9th, and 10th (2 cases) of the month, the four patients having violent diarrhoea and passing much blood. The onset was quite sudden in every case. It was thought that “food poisoning” could be excluded, and the main object in directing attention to the outbreak was to ascertain if any defective drainage existed at the house. Incidentally it may be remarked that the house drainage, etc. had been completely overhauled in 1907 and that no defects existed in February last except one leaky joint on a waste pipe.

The outbreak of food infection which we are about to describe is of interest from more than one point of view. Not only was it possible to determine the organism which caused the outbreak and to trace its source to a paratyphoid carrier, but the bacteriological results yielded an important contribution to the difficult question of the differentiation of Bacillus paratyphosus B. and B. enteritidis, which for some years has been much discussed among bacteriologists.

Ever since artificial media have been used for the cultivation of micro-organisms, the question as to what is the best reaction—that is the degree of acidity or alkalinity—has been a moot point. It has been assumed gratuitously that there is an optimum reaction which is the same for all the common bacteria met with in the laboratory. In recent years however some attempts have been made to settle the question definitely, notably by Eyre and Deelemann. On enquiring what reaction of medium was adopted in six different London laboratories, Eyre found that very divergent standards were used, ranging from 20 units of acidity to 10 units of alkalinity on his scale. (20 units of acidity implies that 20 c.c. of normal alkali must be added to each litre to make it neutral to phenolphthalein. In the case of alkalinity the indicated number of c.c. of normal acid must be added.) The importance of having a definite reaction had until recently not impressed itself upon the majority of bacteriologists. The possibility that although many organisms may grow on media of comparatively widely differing reactions, yet only one of these reactions may be suitable for the growth of a more delicate kind, had been overlooked. Consequently they failed to grow bacteria which by careful adjusting of the reaction of the medium could be readily cultivated. Martin (1910), for example, has lately pointed out the ease with which gonococcus can be made to grow on serum agar if the reaction is carefully adjusted. In later years the subject has assumed still more importance in connection with the production of bacterial toxins, which have been submitted to a large amount of investigation. In the production of diphtheria toxin it was soon recognised that bouillon containing more alkali than is usually employed for ordinary bacteriological work, yielded the most toxin, and even various devices were invented to avoid the initial production of acid which usually takes place as the consequence of bacterial growth in a sugar-containing medium. Madsen (1896) has made a thorough examination of the factors governing the production of diphtheria toxin. By following the reaction of a growing culture from day to day he found two different types of growth occur,—one in which the medium increases in acidity without abatement, and such cultures are atoxic; in the other the primary increase in acidity is followed by a decrease, and subsequent increase in alkalinity. These cultures are sometimes toxic. The two conditions seemed to have relation to the amount of alkali added in the preparation of the bouillon, in that a strongly alkaline medium gave an alkaline end reaction while a less alkaline medium gave sometimes an acid and sometimes an alkaline end reaction.

A short time ago I (1911) described a method for the quantitative estimation of indole. It may be of interest to reproduce the curves obtained by the use of this method, in the study of several strains of Bacterium coli, as curves representing indole-production have seldom been published.

The organisms examined have been killed, (a) by temperatures which should not have destroyed the reducing enzyme, (b) by sodium fluoride and four other antiseptics, derivatives of phenol, and (c) by acetone. In each case there has been total destruction of the reducing power, any apparent production of nitrite from nitrate being too slight to justify the assumption that it is due to anything more than experimental error. Filtrates of fluid cultures have also failed to reduce. The results may be summed up as follows:

(1) The reduction of nitrite by certain dysentery bacilli is not due to a soluble extra-cellular enzyme.

(2) Dysentery bacilli and B. coli killed by a minimal degree of heat have little if any effect in reducing nitrites.

(3) Organisms killed by antiseptics or by acetone do not reduce nitrates and nitrites.

(4) Even where all the organisms are not dead, reduction may be prevented by antiseptics.

(5) The high antiseptic value of tetrachlor-o-biphenol, tetrabrom-o-kresol, p-chlor-m-kresol and hexabrom-dioxy-diphenyl-carbinol has been established for the organisms of the dysentery group.