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 {Reprinted from the Montreal Mbdioal Journal, Sept., 1891.) 
 
 NOTES ON THE BACTERIOLOGICAL STUDY OF 
 
 DIPHTHERIA.* 
 
 By Wyatt Johnston, M.D., Montrbal. 
 
 My ori^nal object in studying oases of diphtheria bacterio- 
 lo^oally was to settle the nature of a series of those doubtful 
 cases in which a diagnosis is practically impossible by tlie ordi- 
 nary means of observation, and where the occurrence of post- 
 diphtherial paralysis or the outbreak of similar or more typical 
 sore throats is the earliest positive proof of the true nature of 
 the case. As I have found it impossible to continue the work 
 on account of the difficulty in obtaining clinical histories of the 
 cases, I wish to publish the results obtained from the examina- 
 tion of another series of cases, most of which were capable of 
 being diagnosed as diphtheria without much difficulty. 
 
 Some time ago I showed before this Society cultures obtained 
 from cases of diphtheria in which the growth had presented the 
 typical characters of the Klebs-Loeffler bacilli. The main char- 
 acteristics of this organism are : 
 
 (1) Rapid growth in serum at blood temperature leading to 
 the formation of well characterized colonies in 16 to 24 hours. 
 
 (2) Peculiarities of structure, especially the presence of in- 
 volution, bacillus forms having clubbed or swollen ends, with 
 granular, unevenly stained protoplasm. 
 
 (H) Toxic effects, producing pseudo-membranous inflamma- 
 
 * Bead before the Medieo-Obirttrgioal Society of Montreal. 
 
'^v 
 
 w 
 
 n t 
 
 t 
 
 tions, followed by characteristic paresis, in cats aud rabbits, and 
 uniformly killing guinea-pigs in two to five days when injected 
 subcutaneously, with production of necrosis, surrounded by 
 local inflammations and oedema at the site of inoculation, and 
 usually associated with more or less marked parenchymatous 
 degenerations and areas of cell-necrosis of the viscera. This 
 condition is distinguished from other forms of experimental 
 septicaemia by the fact that bacteria are absent from the blood 
 and viscera. ' 
 
 These toxic eflects are most striking, and serve to distinguish 
 this organism absolutely from all other species of bacteria. They 
 depend on the fact that the diphtheria bacilli generate an 
 albuminous poison or toxin which, when absorbed into the system, 
 produces fever, cell necrosis and paralysis, which symptoms and 
 lesions can be also brought about by the injection of sterilized 
 cultures containing the toxin alone without any living bacilli. 
 The primary local lesions in diphtheria are probably due both 
 to the toxic action of the poisonous substances evolved and the 
 presence of the bacilli. , 
 
 The disease diphtheria is therefore complex in nature ; the 
 presence of the local exudation of false membrane and the toxic 
 constitutional effects being each a necessary part of all cases of 
 true diphtheria. As the diagnosis is naturally based upon the 
 local inflammatory appearances, and as diphtheria is by far the 
 commonest cause of pseudo-membranous inflammations, the term 
 diphtheritic has come to be applied to all severe inflammations 
 attended with the formation of false membrane and accompanied 
 by necrosis, while the term croupous is applied to milder inflam- 
 mations where there is no necrosis. It should be borne in mind 
 that the terms diphtheritic and croupous are of anatomical and 
 not of aetiological significance, as the neglect of this distinction 
 has led to a great deal of confusion. Diphtheritic inflammation 
 is most often caused by the disease diphtheria, but is not by any 
 means invariably due to this cause, since every sloughing inflam- 
 mation of a mucous surface presents diphtheritic characters and 
 must be called, on anatomical grounds, diphtheritic. On the 
 other hand, croupous inflammations have been shown by Paltauf- 
 
 fn. 
 
 IM^\ i^ 
 
 (H^' f. 
 
J^ 
 
 KoI'ihIco to be sometimes the effect of the action of the Klebs- 
 Loeffier bacilli, so that in this case we have a croupous non-diph- 
 theritic diphtheria. 
 
 To prevent error, the terms diphtheria and diphtheritic should 
 not be considered synonymous ; and since the word diphtheritic 
 has become too firmly established to be dropped from the voca- 
 bulary, it might be well if a suggestion of Dr. MacAUister 
 (Practitioner J June, 1890) were adopted and the word diph- 
 therial used always to express aotiologically in relation to the 
 specific cause diphtheria, applying the term diphtheritic simply 
 in its more general anatomical sense. 
 
 There is evidence to show that cases occur in which an ana- 
 tomically genuine diphtheritic sore throat may be caused by 
 other organisms than the Loeffler bacilli. Roux and Yersin 
 (Pasteur Annates, July 1890) record with great care several 
 such cases where the infection was due apparently to strepto- 
 cocci. Out of 80 cases of angina admitted to the diphtheria 
 wards of the Ghildrens' Hospital, the bacilli were found in 61. 
 Roux and Yersin had no scruples in classing the other 19 cases, 
 in which no bacteria were found, as non-diphtherial angina, 
 A much more remarkable series was that published by Dr. 
 T. M. Frudden, where, in 24 cases of diphtheritic sore throat 
 occurring in children, streptococci were found in 22 and staphy- 
 lococcus aureus in 2, while the Loeffler bacillus was not met 
 with in a single instance. It is important to note that these 
 cases were obtained from an epidemic among children who were 
 inmates of an institution in which scarletina and erysipelas were 
 epidemic at the time. — (Amer. Jour. Med. Sci., May 1889.) 
 
 A subsequent series of 12 cases, all fatal, by the same author 
 (i\r. Y. Medical Record, April 18th, 1891), showed the presence 
 of the virulent Loefiier bacilli in every instance, and a carefully 
 prepared table appended, giving the total number of cases in- 
 vestigated in this manner by various authors up to date, shows 
 that the Loeffler bacilli were found in 307 out of a total of 342 
 examined — about 90 per cent. The suggestion that the cause 
 of diphtheria in America differed from that in Europe had been 
 previously disproved by Prof. W. H. Welch and Dr. A. C. 
 
 >^.^ 
 
 
Iv- 
 
 ti. 
 
 Abbott, who, in a o^t exaot study of eight oases of typical 
 diphtheria ooourring in Baltimore, found the Loeffler bacilli 
 present in every instance. — (Johns Eopkint Ho%p. BiUletiny 
 Jan. 1890.) 
 
 That diphtheritic membrane can be produced amongst other 
 causes by the streptococcus pyogenes is shown by the uniform 
 presence of this organism in the diphtheritic endometritis occur- 
 ring in puerperal fever. The only statements as to the absence 
 of the Loeffler bacilli in diphtheritis of other regions than the 
 throat is that furnished by Paltauf and Kolisko, who were unable 
 to find the bacilli in this condition or in intestinal or cutaneous 
 diphtheritis phageedena. — ( Wiener med. Woehetuehiiftj No. 8, 
 1889.) 
 
 I have tabulated below the oases collected by Dr. Prudden 
 and added my own cases. (I have not included in this table 
 the cases where I was not able to obtwn satisfactory material 
 for exanunation, as 1 found in cases A 2 and A 3 that the bacilli 
 might easily be missed if the secretions only were examined, 
 and although found in portions of actual membrane). 
 
 OBiUTn. 
 
 Babes 
 
 D'Espine 
 
 Ortman 
 
 gpronok 
 
 Kbaz andTenin.... 
 
 Paltauf and Kolisko. 
 
 Zarniko 
 
 Beck 
 
 Sorenaen 
 
 Baoherioh 
 
 Tangl 
 
 Briecn andFraenkel. 
 Prndden (1st series). . 
 Welch and Abbott. . 
 
 Prudden (2nd series) 
 Johnston .. 
 
 RiFIBEHOI. 
 
 Zeitaohrift f . Hygiene. Bd. 6 . . 
 Revue Medicate de la Suisse, Ro- 
 
 mandCflSra.N'o. 1 
 
 Beri. Klin. Woohen.. 1880, No. 10 
 Central, f. Pathol., Bd. 1. p. 218. 
 Annales de L'Institut Pasteur, 
 
 Deo.1888. 
 
 Wiener Klin. Woch., 1889, No. 6 
 In Aug. Dissertation, Kiel, 1889 
 Zeitsehrift f. Hjrgiene, Bd. 8 
 Nordiskt Medicinskt Arohiy, Bd. 
 
 18. No. 26 
 
 dent. f. Baoteriologie, Jan, 'Ji, '90 
 Gent- f. PatholojHe, Bd. 1, p. 796. 
 Berl. Klin. Wochen.. Mar. 17, '90 
 Am. Jour. Med. iSeie.. May 1889. 
 Johns Hopkins Hosp.Bnl., vol. 2, 
 
 N. T. Med,'Reoord, Aprill8ii89i 
 Montreal Med. Jour., Sept. 
 
 No. of 
 
 Cases 
 
 Examined. 
 
 Present 
 in. 
 
 42 
 
 14 
 16 
 
 7 
 
 16 
 SO 
 20 
 52 
 
 10 
 22 
 18 
 22 
 24 
 
 8 
 12 
 iO 
 
 S42 
 
 Loeffler Bacilli. 
 
 42 
 
 14 
 15 
 
 7 
 
 15 
 50 
 18 
 50 
 
 7 
 20 
 18 
 
 8 
 12 
 
 9 
 
 307 
 
 Absent 
 in. 
 
 
 1 
 
 
 
 
 2 
 2 
 
 3 
 2 
 
 
 24 
 
 
 
 1 
 
 35 
 
 1ii H I 
 
 The method of examining is very simple, and no one having 
 an elementary training in bacteriology would have any trouble 
 in carrying it out Following the directions of Boux and Yendn 
 
 ■. ■ "■■;'-*-•> ,\- 
 
 
»f<r#r::'f 
 
 
 
 1 
 
 
 
 
 
 2 
 2 
 
 
 
 
 
 21 
 
 
 
 1 
 
 IT 
 
 {Annalet Pcuteur,, July 1890), I employed sterilized serum, 
 obtained either from ox-blood or from hydrocele or pleuritic 
 exudation. I can quite confirm the statements of these observers 
 as to the advantage of this medium over agar-agar jelly, since 
 by employing serum the colonies of the diphtheria bacilli are 
 readily recognizble at the end of twenty hours, or even earlier 
 if the serum be " improved " in the manner recommended 
 by Loefl9er, through the addition of one-fourth its bulk of a broth 
 containing peptone, beef-tea and sugar. On the other hand, if 
 agar-agar be employed, the colonies are never recognizable 
 before the end of forty-eight hours, and show nothing strikingly 
 characteristic before the fourth day. As the essential object of 
 the examinations is to make an early and positive diagnosis, the 
 saving of twenty-four hours would seem to be of vital importance 
 in itself, but the serum method has also the advantage of per- 
 mitting the diphtheria bacilli to bring their colonies to 
 maturity before the other bacilli which are present have even 
 commenced to form visible colonies. With agar, on the other 
 hand, the two days needed for the appearance of the diphtheria 
 colonies affords ample time for the development of the putrefac- 
 tive forms, if these are present in any large number. The only 
 advantage of the agar method is that the pyogenic staphylococci 
 and streptocacci which are usually present have more character- 
 istic growth than on serum — a matter of secondary importance. 
 There is a current impression that the serum is diflScult and 
 troublesome to prepare, and this has led to its use being avoided 
 in many laboratories when any other medium ';ro be substituted. 
 This idea is quite erroneous, as serum is as eaaiiiy made as any 
 of the other nutrient media — in fact, far easier than gelatine, if 
 prepared according to the method given by Hueppe {Centralh. 
 f. Bact.^ July, 1887), which consists in coagulating and steriliz- 
 ing the serum at once simultaneously. After the tubes are filled 
 to a depth of one to two inches they are lud obliquely in rows 
 in a thermostat, which is then heated till the inner temperature 
 reaches 68^ to 75^G. After half an hour or more at this tem- 
 perature the tubes will be found to have coagulated, leaving the 
 serum nearly transparent. The temperature can now be raised 
 
 s.'^SS^^^^^S^ 
 
w 
 
 %i:' 
 
 rip 
 
 Ny 
 
 to about 90° by bringing the water in tho jacket to the boil, 
 and the tubes should be exposed to this heat for half an hour 
 each day on three or four successive days, when, on placing them 
 in the incubator in the usual manner, all but a few will be found 
 to remain perfectly sterile. If Loeffler's serum is required, the 
 serum is mixed with one-fourth its volume of LoefHer's bouillon 
 before filling into the tubes. This addition does not interfere 
 with the property of coagulating and remaining transparent. 
 If blood serum is employed, care should be taken in collecting 
 it that the clot is allowed to form before transporting the jars of 
 blood. After standing forty-eight hours in a refrigerator or in 
 a cool cellar, an abundant supply of clear serum can be obtained. 
 The presence of small traces of haemoglobin i)i the serum does 
 not much impair its translucency, certainly not enough to render 
 it unsuitable for the isolation of diphtheria bacilli. 
 
 The serum tubes could be prepared and kept in stock by 
 druggists if the method ever comes into general use, which seems 
 unlikely. The examination may be made from material taken 
 direct from the throat by scraping the membrane, or, preferably, 
 a piece of membrane maybe detached by a pair of forceps or a 
 swab of cotton wool. If the membrane has to be transported, it 
 may be put into a clean, dry test-tube or folded up in blotting 
 paper. To examine, it need only be moistened by a drop 
 of sterilized water. A microscopical examination can be made 
 by smearing the piece of membrane over the surface of a cover- 
 glass, passing it three times through a flame. After drying 
 and staining with a drop of any aniline stain, but preferably by 
 Gram's method, the diphtheritic bacilli are seen as short thick 
 rods (about the same length as tubercle bacilli), lying in little 
 groups. These bacilli are present in enormous numbers in the 
 early stages of diphtheria, but diminish rapidly in number as 
 the membrane softens. The highly characteristic involution 
 forms, which assume comma or club shapes with swollen ends, 
 and present a protoplasm broken up into small granules, are only 
 recognizable with a good immersion lens. 
 
 The cultures are made by drawing the infected needle in 
 parallel lines along the surface of the serum. By treating two 
 
 mmpp 
 
 *>..■■ 
 
 
 .%.> 4 , 
 

 made 
 
 cover- 
 
 rying 
 
 lyby 
 
 thick 
 
 little 
 in the 
 iber as 
 olution 
 
 ends, 
 re only 
 
 edle in 
 ing two 
 
 or three tubes in this fashion, the infecting material at Brat 
 abundant and producing a continuous dense growth, will in the 
 second or third tube only implant a very few bacteria, so that 
 the colonies can be studied separately. The tubes are to be 
 kept at about body temnorature for twenty-four hours, when 
 minute white ()oints appear on the surface of the serum, attain- 
 ing about the size of pin-heads or of split pens. These, if found 
 under the microscope to consist of bacilli, are probably the diph- 
 theria bacilli, as the other forms would not have attained such 
 size in so short a time. 
 
 The other colonies which attain such proportions in twenty- 
 four hours are almost invariably found to be micrococci, usually 
 the staphylococcus pyogenes. 
 
 I will not go further into the culture experiences with the 
 bacillus than to say that I have been able, in all my cases, to 
 confirm the statement first made by Welch and / Sbott, of the 
 Hygienic Institute, that the bacilli form an abundant invisible 
 growth on potatoes, a medium stated by previous observers to 
 be unsuitable for its cultivation. I also can substantiate the 
 statement of Beck, tliat the agar cultures do not show the char- 
 acteristic involution forms. 
 
 The method, however, has a serious defect, since an organism 
 exists which is identical in size and appearance with the Loeffler 
 bacillus, and grows on serum in a similar manner, though totally 
 devoid of pathogenic properties. This is called the pseudo- 
 diphtheria bacillus, and has been frequently found in the throats 
 of healthy persons, as well as in follicular tonsillitis. The proof, 
 therefore, is not absolute until substantiated by the inoculation 
 of some susceptible animal. How far this pseudo-diphtheria 
 bacillus would interfere with the method in practical work is 
 not yet certain. Personally I have so far only met with the 
 virulent or true bacillus. 
 
 The results of my examinations are as follows : — 
 
 Of nine cases examined in which the diagnosis of diphtheria 
 could be made without much difficulty from the symptoms and 
 the appearance of the throat, the Klebs<Loeffler bacillus was 
 found in eight. In the case where it was not found, the con- 
 
 i%.^ ^"'« 
 
 ■li.*'. 
 
I 
 
 I 
 
 dition was one of an undoubted false membrane which contained, 
 microscopically, large numbers of bacilli which appeared to be 
 the organisms in question but did not appear in the cultures. 
 When the specimen was taken the throat had just been freely 
 sprayed with a solution of hydrogen peroxide, and the nega- 
 tive result may have been due to its disinfectant power of 
 inhibiting the growth, though the fact that colonies of staphylo- 
 coccus aureus appeared make that less likely. There was an 
 anomalous course in this case, since the throat was found per- 
 fectly clear of membrane on the following day, preventing my 
 repeating the experiment. In spite of this the disease appears 
 to have been true diphtheria, as the nurse stated that the patient 
 had a distinctly nasal voice when he left the hospital, ten days 
 later. The bacillus was also found in an anomalous case where 
 an extensive false membrane existed with almost no disturbance 
 of the general health. In this case there was no paralysis. 
 
 In six cases where the diagnosis was doubtful, the bacilli were 
 not found. Several cases of follicular tonsillitis and a case of 
 scarletinal sore throat were examined with negative results. 
 
 I have divided the cases into two groups — (a) where the con- 
 dition was clinically like diphtheria, and (b) anomalous cases. 
 The cases are as follows, the first being given in detail and the 
 remainder summarised in a table at the end of the article. 
 
 Case 1. — M. L., female, aged 21, admitted April 9th, 1891, 
 into the female medical ward of the Montreal General Hospital, 
 under Dr. Ross, with a suspicious-looking patch of membrane in 
 both ton^s and a tiny membranous patch on the side of the 
 uvula ; temperature 102^^. Next day an extensive patch of 
 dirty gray membrane was seen in the uvula and soft palate. 
 Transferred to diphtheria ward. Seen on April 11th ; tempera- 
 ture 100^ ; had a patch of membrane on left anterior pillar of 
 fauces. Discharged May 2nd ; no paralysis. 
 
 Microscopic examination of the membrane showed an enor- 
 mous number of short, thick bacilli, a few larger bacilli, and 
 some clusters of micrococci. 
 
 Cultures in serum made on May 11th and kept at 35^C. 
 showed on the following day numerous small, flat, white colonies 
 
 ti:. 
 
Ln enor- 
 [iUi, and 
 
 It 35«'C. 
 I colonies 
 
 1 
 
 composed of short, thick hacilli, having the typical appearance 
 of the Loeffler bacilli. Involution forms numerous. In agar 
 plates small, flat colonies appeared on the second and third 
 days, showing superficial thin cono^^ittrio growjth from a small 
 central white spot. In stale culture in agar the growth, on first 
 transplantation, was barely visible, but in subsequent generations 
 the colonies became more distinct, forming always flat, circular, 
 concentric, white growths on surface about the entry point of 
 the needle. Microscopically the bacilli in the agar cultures 
 were short, thick rods with rounded ends ; their length was 
 about 0.0015 to 0.002 mm. (one and a hall' to two-thousandths 
 of a millimetre). None of the agar cultures showed involution 
 forms. 
 
 April 2Sth, 1891. — Inoculated a very large, full-grown 
 female guinea-pig with 0.7 com. of a watery suspension obtained 
 from washing a seven days old culture in six per cent, glycerine 
 agar-agar. Injected subcutaneously in right flank. 
 
 May nth. — Found dead in cage ; had been quiet and refused 
 food for past three days. In right flank, at site of inoculation, 
 induration and gray opacity of skin and subcutaneous tissue, 
 with some hemorrhage ; marked oedema extending from this 
 spot to right axilla and backward to right groin. 
 
 Microscopically, a few bacilli corresponding in size to Loefiler 
 bacilli found in the oedematous fluid. Microscopic examination 
 of blood, lungs, liver, kidneys and spleen negative. Kidneys 
 swollen, opaque and grayish in sections ; extensive swelling and 
 degeneration of epithelium in convoluted tubes ; a few casts seen 
 in the tubules. From oedematous fluid cultures in two serum 
 tubes showed on following day numerous small colonies of a 
 bacillus corresponding to Loeffler bacillus with well marked in- 
 volution forms, staining well by Oram's method. Cultures from 
 blood, spleen, lungs, liver and kidneys, all remained sterile. 
 
 The remaining cases, except a few of special interest, are 
 briefly summarised in a table, as they were for the most part 
 practically repetitions of the one given above. One case was of 
 special interest and occurred in the private practice of Dr. Major. 
 In this case, which I saw for the first time on the seventh day, 
 
 * > 
 
 
 .i«^4«. 
 
10 
 
 I..: 
 
 i 
 
 fl/^ 
 
 the membrane had nearly all gone, and on the first examination 
 no Loeffler bacilli were found, although two agar tubes and two 
 serum tubes, as well as three Petri dishes, were all seeded from 
 a piece of membrane. In this case an oidium appeared strongly 
 resembling that of thrush, and I considered the case to be thrush 
 complicated with staphylococcus infection. Dr. Major stated, 
 however, that at the onset the condition was unmistakeably that 
 of diphtheria, and the correctness of his diagnosis was proved 
 some days later by the onset of severe and persistent paralysis 
 of the palate. In the interval I had obtained a fresh piece of 
 membrane which yielded two colonies of the Loefller bacillus. 
 This case shows that a negative result is of no diagnostic value 
 when the membrane is clearing, even when a fairly exhaustive 
 examination hau apparently been made. In another case I found 
 an oidium growth, readily distinguishable, however, from thrush, 
 associated with a large number of Loeffler bacilli. 
 
 The uncertainty of examinations made at a late period in 
 cases going on to recovery was shown in another case, for the 
 opportunity of examining which I am indebted to Dr. W. S. 
 England. In this case I saw the patient on the seventh day. 
 A distinctly membranous exudation had been present, returning 
 within twenty-four hours after being scraped off, but always 
 confined to the tonsils. In this case smear cultures on five 
 glycerine agar tubes failed to show any bacilli, the seeding being 
 done directly from the membrane in the throat. In this case a 
 tiny particle of membrane which had been obtained at the time 
 of examination was seeded on serum a week later, and yielded 
 two colonies corresponding to the Loeffler bacilli, one of which 
 was tested and found to kill a guinea-pig in the typical manner. 
 
 In two cases I had great difficulty in obtaining suitable ma- 
 terial for examination, owing to the afi^ection being confined to 
 the posterior nares, and where a pi'olonged local examination 
 seemed unjustifiable owing to the profound exhaustion of the 
 patient. In these cases I made cultures daily from the nasal 
 discharge for several days, but without in any case obtaining 
 the Loeffler colonies. In one of these cases (Case A 2) the 
 nurse afterward obtained for me a small fragment of membrane 
 
 :v*w^ 
 
 't''M-M 
 
.%^ 
 
 ition 
 
 two 
 
 from 
 
 irush 
 ;ate(l, 
 J that 
 roved ^ 
 •alys'w 
 ece of 
 iciUus. 
 I value 
 lUBtive 
 [ found 
 thru8h, 
 
 eriod in 
 for the 
 . W. S. 
 ith day. 
 eturning 
 always 
 on five 
 ng being 
 ib case a 
 the time 
 yielded 
 of which 
 manner, 
 table ma- 
 nfined to 
 amination 
 ion of the 
 the nasal 
 obtaining 
 A 2) the 
 lembrane 
 
 11 
 
 from which I obtained colonies having the characteristic culture 
 appearances, and which killed a guinea-pig in the typical manner. 
 In the other case (Case B 2), staphylococcus aureus and citrous 
 were present in enormous number, together with a few strepto- 
 cocci, but no Loeffler bacilli were found. 
 
 In a fatal case (Case A 9), where an autopsy was performed 
 by Dr. Finley, who kindly sent me the respiratory organs for 
 examination, the larynx and trachea showed an extensive 
 sheathing of diphtheritic membrane extending down to the main 
 bronchi. Large numbers of the Loeffler bacilli were obtained 
 from this membrane, and an area of pneumonia from the lung 
 showed a small number of the bacilli associated with a large 
 number of small diplococci. No streptococci were found. 
 
 In an anomalous case already cited, brought to my notice by 
 Dr. H. S. Birkett (Case B 5), an extensive soft, yellowish 
 membrane covered the posterior nares and extended over the 
 epiglottis and into the larynx. This membrane could be readily 
 removed without causing bleeding, but tended to recur. The 
 general health of the child was unaffected. Cultures on agar 
 yielded an abundant growth of a single bacillus form, forming 
 prominent yellow white surface colonies. Inoculated into the 
 conjunctiva of rabbits, no effect was produced, and I was inclined 
 at the time to regard the bacteria as being possibly the pseudo- 
 bacillus, but on re-investigating the cultures some three months 
 later I found that they gave the typical Loeffler colonies on 
 serum, with an abundant invisible growth on potatoes, and showed 
 on both these media most characteristic involution forms. Inocu- 
 lated into a guinea-pig, this organism showed a high degree of 
 toxic virulence, killing the animal in thirty-six hours. The 
 autopsy on this animal showed an opaque hsemorrhagic, reddish- 
 gray indurater area at the spot of inoculation, surrounded by a 
 zone of serous oedema. Cultures from the organs and from the 
 serous exudation remained sterile, but typical Loeffler colonies 
 were obtained from the circumscribed hsemorrhagic patch at the 
 site of inoculation. These colonies, on being transplanted on 
 agar, gave only the characteristic, flat, compact, concentric sur- 
 face growth of the Loeffler organism, and not the abundant 
 
 ^^imm^mf^w^ 
 
 ser(tm^m»m_ 
 
 
 
 
 KW- 
 
i;>i! 
 
 yf 
 
 12 
 
 « 
 
 prominent growth of the original tubes. For this reason it 
 appears probable that the original colonies were impure, contain* 
 ing both the Loeffler colonies and some other form, which latter 
 wad subsequently eliminated in passing through the serum 
 culture. 
 
 In cases which were examined at an early stage the Loefiler 
 bacilli were found almost in pure culture. In most cases the 
 staphylococcus aureus was found, but it was usually scanty. 
 The colonies closely resemble those of the Loeffier bacilli for the 
 first day, but can be distinguished at the end of forty-eight hours. 
 Streptococci were seldom met with, and then only in isolated 
 colonies, the scarcity of this organism being contrary to what I 
 had been led to expect from a study of the literature. In the 
 anomalous or doubtful cases, on the other hand, either staphylo- 
 coccus and streptococcus, or both together, were present abun- 
 dantly in all instances. The influence of these pyogenic bac- 
 teria on the course of true diphtheria is a pomt greatly needing 
 investigation. 
 
 In cases examined during the period when the membrane had 
 begun to soften — that is to say, after the third day in ordinary 
 mild cases — the falling off in the number of diphtheria bacilli 
 was most marked. This did not seem to be due here to the 
 antagonistic action of saprophytic bacteria, as has been commonly 
 assumed, since, as a rule, the cultures made in late stages in 
 cases going on to recovery remained perfectly sterile, except 
 for the few scattered colonies of Loeffler bacilli. This result I 
 attribute to the inhibitory action of the local disinfectant appli- 
 cations — in most of the cases a spray of peroxide of hydrogen 
 was employed — but I have made no experiments to determine 
 this point. 
 
 A matter of considerable interest is the question whether this 
 method uf examination will ever come into general use, and if 
 such be the case, what class of practitioners could carry it out 
 to the best advantage. This point is considered by Prof. Welch 
 of Baltimore in an address delivered before the ninety-third 
 annual session of the Medical and Ghirurgical State Faculty of 
 Maryland in April, 1891 (^Medical New», May 16, 1891). In 
 
 "Sf-j' 
 
13 
 
 )on it 
 ntain- 
 latter 
 aerum 
 
 ioeffler 
 es the 
 icanty. » 
 for the 
 ; hours. 
 Bolated 
 what I 
 In the 
 ;aphylo- 
 it abun- 
 nic bac- 
 needing 
 
 ane had 
 ordinary 
 ia bacilli 
 e to the 
 )mmonly 
 (tages in 
 (, except 
 , result I 
 int appli- 
 lydrogen 
 letermine 
 
 jther this 
 se, and if 
 rry it out 
 3f. Welch 
 aety-third 
 jaculty of 
 |91). In 
 
 this a(^<l- , which by a masterly treatment of the facts places . 
 the prop ji&xK and treatment of diphtheria on a broad scientific 
 basis, Professor Welch does not think that the hopes of Roux and 
 Yersin, that the method may yield good results in the hands of 
 unskilled persons, are likely to be realised. He thinks that an 
 elementary training in bacteriology is needed in those carrying 
 it out, and suggests that as elementary courses on bacteriology 
 have now become so common that it will not be long before most 
 communities will possess at least one person capable of doing the 
 work satisfactorily. My own experience has shown me that 
 doubtful cases, which are those in which the value of the method 
 should be best shown, the chief difficulty is in obtaining suitable 
 material for examination are of two kinds — first, those where 
 the condition resembles tonsillitis and the exudation is confined 
 to the tonsils ; and, second, those where the local disease is 
 situated in some part of the respiratory tract not readily exam- 
 ined, or where the severity of the constitutional symptoms ren- 
 ders a prolonged examination difficult. As the procuring of a 
 small piece of the false membrane at the earliest period possible 
 is the sine qua non, and to do that in this latter class of cases 
 requires a special manipulative skill only possessed by a skilled 
 laryngologist, the task will properly fall to this individual. The 
 aid of a throat specialist seems indispenable in cases where the 
 local examination presents much difficulty. Possessed of suitable 
 material, the best results in the further examination would cer- 
 tainly be obtained in a properly equipped laboratory. 
 
 In the cases where the confusion arises from the membrane 
 being confined to the tonsils, the case is much more simple, and 
 there is less need of the services of the laryngologist; all 
 that is required being to detach & small piece of the exudation, 
 wrap it in paper, and send it to some laboratory for examination. 
 Considering the gravity of the interests at stake in the prompt 
 recognition and isolation of cases of diphtheria, one would natu- 
 rally suppose that a method which enabled a positive diagnosis 
 to be made within twenty-four hours, in cases seen during the 
 first days of the illness, would be welcomed eagerly by the pro- 
 fession. That this has not happened is due probably in great part 
 
 
 
 
 
 i|V?.;«!0? 
 
 ''-i^^ 
 
 mm 
 
 V 
 
 
 ^^^itf 
 
 mi'"-' 
 
 .S-' 
 
 h& 
 
 i, .'■.4 
 
^?* 
 
 fh! 
 
 I 
 
 14 
 
 to tho wise precaution of treating all doubtful cases as if they 
 were cases of diphtheria, and possibly, too, in some degree to 
 a tendency to pride ourselves upon our sagacity, valuing the 
 result of a clever guess more than that obtained by a less 
 brilliant, though more certain, method. As a matter of experi- 
 ence, a large proportion of the doubtful cases, especially the 
 tonsillitid group, declare themselves to be one thing or the other 
 by the time the doctor makes his visit on the following day. 
 in conclusion, it may be stated ; 
 
 (1) That in almost all cases where strong clinical grounds 
 exist for the diagnosis of diphtheria, the bacteriological examina- 
 tion has shown the almost invariable presence of the malig- 
 nant Loeffler bacilli. 
 
 (2) That, excepting in connection with soarletina, measles or 
 erysipelas, the number of cases of diphtheritic sore throat due 
 to other causes is very small. 
 
 (3) That in doubtful cases the accuracy of the method de- 
 pends chiefly upon obtaining suitable material at an early stage 
 of the disease. i 
 
 (4) That the method is not of much service in doubtful cases 
 where the difficulty is due to the infection occurring in localities 
 difficult- to examine without skilled manipulation, unless suitable 
 material is obtained for examination. 
 
 Summary op Method for Detecting Loiffler Bacilli. 
 
 Microscopical Exam'maiion. — Stain a cover-glass suieared by a bit of 
 mem braue with any' aniline dye. The bacilli are arranged in small 
 clumps, and are short, thick roils, about same length as tubercle bacili, 
 but much thicker ; numerous beaded and drumstick shui)es met with 
 —in solution forms- Gram's staining method 'jau be employed. 
 
 CtUiures. — Can be made direct from membrane in throat or from 
 ^<mull bit of membrane folded dry in clean paper, iNo special antisep- 
 tic precautions necessary. Toucli or scrai^e membrane with a sterilized 
 platinum needle and draw it in parallel streaks over the surface of a 
 seruni tube, using two or more successive tubes before reinfecting the 
 needle. Keep the tubes at body temperature. In 20-24 hours the 
 Ld'tiler bacilli appear as small grayish-white points, size of pin-heads, 
 showing under the microscope the charat^teristic ap|jearances of the 
 bacilli in the original membrane. 
 
 Diagnom. — (o) (^ther bacilli do not form visible colonies at twenty- 
 four hours, {h) Btapiiylococcus colonies resemble those of La>ftler 
 bacilli to naked eye apj)earance, but recognized on microscopic 6xami- 
 nation. (c) Pseudo-diphtheritic bacilli have microscopic and culture 
 characters of the La-tiler bacilli, but have no pathogenic properties. 
 
^ g^T:^ 
 
 '^^mit:: 
 
 16 
 
 Pathogenein$.—fixihcvttmeonB inoculation of guinea-pig kills in two 
 to five (lays, witli lucniorrhagic necrosis and uHlema at site of inocula- 
 tion. Tlie bacilli can be recognisevi microscopically and by culture 
 near spot of inoculation, but blood and viscera give negative results. 
 Disseminated parenchymatous degeneration of liver and kidneys. 
 
 The following is a tabular analysin of the cases, divided into 
 
 two groups : — 
 
 Groui- a.— Cases evidently diphtheria. 
 Number of cases examined, 9. Result : Positive H ; Negative, 1. 
 
 No. 
 
 Name. 
 M. L. 
 
 Sex. 
 
 Age 
 
 Service. 
 
 Course. 
 
 Result of 
 Examination. 
 
 Remarks. 
 
 1 
 
 F 
 
 21 
 
 M. a. Hospital. 
 
 Recovery 
 
 Typical.* Oidium 
 also found- 
 
 Bexan as Ton- 
 sillitis, 
 
 2 
 
 l;b. 
 
 M 
 
 2 
 
 Do. 
 
 Death. 
 
 Typical. 
 
 Nasal diph- 
 theria. Ba- 
 cilli in bit of 
 m e m b nine. 
 None in 
 nasal secre- 
 tion. 
 
 3 
 
 Mrs.T. 
 
 F 
 
 2& 
 
 Dr. England. 
 
 Recovery 
 
 Two colonies only. 
 
 
 4 
 
 W. P. 
 
 M 
 
 16 
 
 M. U. IIo8]>ital. 
 
 Do. 
 
 ^^o Luffler bacilli ; 
 abundant stapby- 
 loouccus pyogenes 
 aureus. 
 
 Rapid disap- 
 pearance of 
 iuombrane, 
 (nasal voice). 
 
 5 
 
 L. M. 
 
 F 
 
 10 
 
 Dr. Major. 
 
 Do. 
 
 Only two colonies 
 found. Oidium 
 present 
 
 Marked diph- 
 therial para- 
 lysis. 
 
 6 
 
 F. P. 
 
 M 
 
 8 
 
 M. G. Hospital. 
 
 Do. 
 
 Typical. . 
 
 
 7 
 
 E. G. 
 
 F 
 
 10 
 
 Do. 
 
 Do. 
 
 Typical. 
 
 
 8 
 
 L. P. 
 
 M 
 
 13 
 
 Do. 
 
 Do. 
 
 Typical. 
 
 
 9 
 
 M.O. 
 
 F 
 
 4 
 
 Do. 
 
 Death. 
 
 Typical. 
 
 
 * In cases marked "typical " a large number of the virulent La>tiier bacilli were 
 found. 
 
 G.oupB. — Anomalous cases.^' 
 Number of cases, 5. Kesult : Positive, 1 ; Negative, 4. 
 
 1 
 
 O.P. 
 
 F 
 
 20 
 
 M. a. Hospital. 
 
 Recovery 
 
 Negative. 
 
 Exudate con- 
 fined to Ton- 
 sils. 
 
 2 
 
 e.w. 
 
 P 
 
 24 
 
 Do. 
 
 Do. 
 
 Negative. 
 
 Nasal diph- 
 theria? 
 Nasal voice. 
 Local exam- 
 ination un- 
 satisfactory. 
 
 3 
 
 A.C. 
 
 M 
 
 1 
 
 Do. 
 
 Do. 
 
 Negative. 
 
 Local exam- 
 ination un- 
 satisfactory. 
 
 5 
 
 W.J. 
 
 M 
 
 8 
 
 Do. 
 
 Do. 
 
 Negative. 
 
 Scarletina. 
 
 6 
 
 s. 
 
 M 
 
 8 
 
 Dr. Birkett. 
 
 Do. 
 
 Positive. 
 
 
 * Cases 1, 2 and 3 ol this series were placed in the diphtheria ward of the Montreal 
 Oeneral Hospital. Several oases of follicular tonsillitis were also examined, always 
 with negative results.