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TWO NEW FRENCH METHODS FOR STAINING BLOOD FILMS AND BLOOD PARASITES.
142 that other
ampoules
from the
same
box
were
used
on
the
surgical side without success. The failures occurred in groups of 3 and 4, and we believe that most, if not all of them, were due to defective solutions. A few drops of Partial failure occurred in 15 cases. chloroform was all that was necessary to finish the operation.
TWO NEW FRENCH METHODS FOR STAINING BLOOD FILMS AND BLOOD PARASITES.
BY L. TRIBONDEAU. We have therefore had about 1’28 per cent. complete MEDICAL OFFICER OF THE FRENCH NAVY. failures and 0 90 per cent. partial failures. There were no deaths from stovaine. THESE two methods, now brought before the readers of Course and extent of ttzv analgesia.-The course of the THE LAN.CET. are very practical, not only because the analgesia varied with amount injected and place of in- technique of the colouration is simple, but especially because after the sensation injection perineal jection. Immediately the staining solutions can be prepared, without difficulty by In about 3-5 minutes (in rare cases 8-10) was abolished. anybody. They are related to the well-known methods of the analgesia would have gradually extended up to the Leishman and provide efficient substitutes for the secret umbilical region if injection was made in fourth Illmbar space of Giemsa and other German writers. and to the ensiform cartilage if in the third. In a few cases processes I. Colouration Method with Stain III. sensibility to heat was retained for some time after complete Of the two methods, this one (neutral solution of eosinate analgesia had been established. Accompanying symptoms.-In a small proportion of cases of methylene-blue converted by ammonia) is more rapid in (less than 2 per cent.) nausea occurred. Actual. vomiting execution and gives more complete results, but it requires occurred in about 3 per cent., bnt never lasted more than a very pure and neutral distilled water. Certain commercial minute. Relaxation of sphincter during analgesia with incon- distilled waters do not possess these qualities.2 Preparation of Stain III.-Heat some distilled water to tinence of faeces occurred in a few cases. Signs of depression, such as yawning, pallor of face, and perspiration, rarely boiling point. Divide. (A)-50 c.cm. in enamelled basin ; add 0-20 g. pure medicinal methyoccurred and have seldom caused anxiety. Pulse at end of blue ; shake to dissolve. operation was in many cases stronger than at beginning. (B)-75 c.cm. in a glass; add 0.30 g. water-soluble postn (French shake to dissolve. Post-operative symptoms.-In about 1 per cent. of the cases Pour B into A by successive fractions. After each addition a certain amount of headache, easily amenable to treatment, of B mix for some time with glass rod ; then place on glass was complained of. The temperature rose a degree or slide drop of mixture from end of rod. This drop is at first two on second and third days in some of onr early cases. i deep blue and free from precipitate. Then, as proportion of Vomiting after operation was conspicuous by its entire eosin is increased, the blue becomes more pale and a preabsence. Paralytic sequences, whether transitory or per- ’, cipitate appears. Finally precipitate increases and colour of have never occurred. manent, liquid turns from blue to rose. Stop addition of eosin as soon as this change occurs. It generally requires a little The Solution Used. more than 50 c.cm. of B to obtain this result, which is quite Stovaine billon was used in about half the cases. Each easy to observe. Add 4 of ammonia to mixture thus obtained. Mix. ampoule contained a little over 2 c.cm. of the following: Heat toc.cm. 120° C. ’in autoclave for 20 minutes in enamelled Borate of epirenin, 000013; stovaine, 0-04; sod. chlor., basin covered with inverted glass funnel. Remove from 0 -0022 ; distilled water to 1-00. autoclave ; stir with rod and allow to cool completely. The amount injected is the same for a big or small operaFilter through small white filter-paper well folded all tion-viz., 2 c.cm. of the solution containing 0-08. In some contents of basin. Discard filtrate. only precipitate, which is almost entirely retained on filter, and of which a cases we injected half or three-quarters of the dose, but the effect was not quite so good and passed off more quickly. small portion remains deposited on side of basin. Dry preAs an exception we may mention colporrhaphies, in which cipitate by placing in incubator at 37° C. the filter widely on several layers of absorbent paper, and also basin. half a dose is sufficient,’bnt even there it is better to inject open When drying is complete (no trace of water must remain a full dose, as it may be found necessary to do something in order that ammonia may be completely volatilised) place more. filter in basin and dissolve as much as possible of dried stain We are now using with even better results a solution by pouring into basin, in successive fractions, 100 c.cm. of (absolute ethyl alcohol, 90 c.cm.; neutral prepared in the school chemical laboratories under the glycerinated alcohol 10 c.cm.) and crushing powder with large glass rod. supervision of Dr. Bahry Bey. The formula used is: glycerine Sod. chlor., 0 °0011 ; stovaine, 0 04 ; water, 1 c.cm. It is Transfer the 100 c.cm. of solution into flask, taking care to transfer also all undissolved stain. Shake flask from time prepared as follows :to time. Filter after 12 to 24 hours. The sodium chloride is dissolved in the water. The soluTechnique of staining.-First fix preparation with undiluted tion is sterilised in the autoclave for 15 minutes and then Stain III. For this, slide carrying blood (previously spread left to cool down. The stovaine is dissolved in solution and in thin film, dried simply by moving in air, and marked off filtered through a good filter previously sterilised. It is by a glass pencil line) is placed on table, film upwards. then heated on a water-bath for ten minutes at 80°-90°C. Cover film with 0’2 c.cm. of Stain III. (approximately After cooling it is stored in sterilised ampoules. The whole 12 drops). Cover with half of Petri dish to prevent too great Allow to act three process is carried on aseptically. Attempts to sterilise the evaporation, especially in summer. stovaine solution by boiling have resulted in the decomposi- i minutes. Then stain by adding to Stain III., on slide itself, 0’6 c.cm. tion of the stovaine and is not necessary, stovaine itself being i ’ of distilled water (approximately 12 drops). Mix water and antiseptic. stain by few movements of slide. Replace on table. Allow Conclusions. to act for average time of 12 minutes without moving. Wash Stovaine analgesia was greatly welcomed by our patients. rapidly with jet of distilled water. Dry immediately (by women have a Most Egyptian great dislike to general anves- passing wet preparation for two seconds over flame and thesia. Many instances have occurred of patients refusing blowing vigorously on it). to have an operation done under chloroform, but agreeing to II. Colouration Method with Stains I. and II. undergo the operation under stovaine analgesia. When good distilled water is not available this second The preparations are made and the stovaine injected before the students come into the theatre. The patient is covered method (neutral solution of eosinate of natural methylene blue alkaline solution of eosinate of methylene blue converted by up with sterilised towels, and is less exposed than during an and ammonia) is preferable to first; it is less delicate. examination in the out-patient department. After having watched this number of patients during their Preparation of Stain L-Pour into heat-resisting flaskNeutral glycerine 5 c.cm. operations and convalescence, and having followed many of 45 c.cm. Absolute ethyl alcohol them for years afterwards, we have no hesitation in asserting Pure medicinal methylene blue............ 0*20 g. that stovaine is, in our opinion, far superior to chloroform in Water soluble eosin (French) 0’05g. all gynaecological operations. During the operation the Dissolve rapidly by plunging flask in very hot-water bath complete relaxation of abdominal muscles and the evenness! and shaking. Allow to cool. Pour into glass-graduated of the analgesia are a great help to the operator. The 1 C. R. Soc. de Biologie de Paris, May-June, 1918. absence of vomiting during the convalescence prevents any One can correct a defective water by redistilling, after addition of strain on the stitches. The freedom from shock and all a 2little silver carbonate obtained by precipitation of a silver nitrate solution with carbonate of soda, but this is complicated. respiratory troubles are points in favour of stovaine.
lene eosin);
’
even
Keep
.....................
..................
............
143 measure; make up to 50 c.cm. with absolute ethyl alcohol. Filter and cork in a flask. Preparation of Stain II.-Pour into heat-resisting flaskNeutral glycerine Ethyl alcohol at 95 per cent............. Pure medicinal methylene blue Water-soluble eosin (French)
.....................
............ ............
25 c.cm. 15 c.cm. 0’20 g.
0’05g.
(c) Age ot’ specimen.-The stool must be cultured in a fresh state, as the bacilli of dysentery ouickly die off in a stool_ If the sample cannot be obtained at a convenient time it should be placed in an ice-chest. A fresh specimen from a case on second day of disease was examined and B. dysenteriæ Shiga was isolated in almost pure culture. Another specimen from the same case was examined as follows :Fresh specimen (fifth day of disease) plated immediately, 27 colonies
Dissolve in water bath as with I. Allow to cool. Pour into glass-graduated measure, and make up to 40 c.cm. with of B. Shiga. 95 per cent. ethyl alcohol. Return into flask. The specimens plated after standing at room temperature or in iceAdd 4 c.cm. of ammonia. Mix. Heat to 120° C. in autoclave for 20 minutes in open flask. Remove from auto- chest gave the number of colonies of B. Shiga stated:Pour into glassclave and allow to cool somewhat. graduated measure and make up to 50 c.cm. with 95 per cent. ethyl alcohol. Return into flask, which is corked only after a dav or two. Teclzrnique of staining.-First fix preparation with unslide on table, film upwards. diluted Stain 1. For this, Cover film with Stain I. Cover with Petri dish. Allow to A fresh specimen from an acute case was cultured, and B. dysenteriæ act three minutes. Wash with jet of distilled water. Get Flexner was isolated easily, the majority of colonies on the plate being rid of surplus water by shaking; wipe under side of slide; of this organism. place it on edge of glass or crystallising dish without The day after a six hours’ old sample from the same case, with film. similar naked-eye and microscopical appearances, was cultured. Not Stain by pouring on preparation Stain II. diluted and hot. one colony of B. dysenteriæ was present on the plate. For this have small test-tube of 1 em. diameter marked at These are most striking facts which prove the absolute 2 c.cm. ; pour distilled water to mark; heat tube held aslant of culturmg fresh specimens. We have recorded in flame until appearance of first bubbles of air ; add to the necessity similar examples. 2 c.cm. of hot water 4 to 5 drops of Stain II. ; pour hot over many It is recommended that the MacConkey’s medium used be film. Allow to act 15 minutes. Wash with jet of distilled water. Dry immediately (heating I made up with double the strength ofneutral red-i.e., and blowing). Remove excess of blue by pouring on dry 0’5 c.cm. of a 1 per cent. solution to every 100 c.cm. of agar. preparation held aslant watery solution of tannin (1 in 20) lYlethod of Plating. until film becomes rose-coloured. (It is necessary to avoid differentiating moist films because preparations would With sterilised platinum loop pick from freah (a) become spotted with blue. The solution of tannin is pre- specimen of fsecea a small portion consisting of pus and pared by dissolving 1 g. of tannin " a 1’alcool" in 20 c.cm. of mucus. very hot water. One adds a little camphor to prevent growth (b) Wash it well in sterilised normal saline and remove Wash at once with distilled water. of moulds.) Dry it immediately with loop, allowing immediately. excess saline to drain away along N.B.-One can omit I., fixing simply with alcohol, but it is sides of tube. then necessary to stain with II. for 30 minutes. (c) Rub and spread washed pus and mucus well on to surface of MacConkey plate nt position (A) shown in diagram. THE ISOLATION OF DYSENTERY BACILLI (cl) Sterilise loop and make vertical stroke (B) from first inocuFROM THE FÆCES.
place
drying
lation.
BY HENRY
WHITEHEAD, M.D., D.P.H. MANCH., M,B., B.S. LOND.,
TEMPORARY CAPTAIN, R.A.M.C. ; BACTERIOLOGIST TO — GENERAL
HOSPITAL; AND
J. KIRKPATRICK, LANCE-CORPORAL, R.A.M.C.
(T.F.).
OUR object is to describe what we consider the best method of isolating B. dysenteriæ from the fxces. Collection and Seleotion of Fæces for Examination. (a) Duration oj disease.-Specimen must be obtained during and as early as possible in acute stage of illness. After a week isolation of B. dysenteriæ becomes increasingly difficult, and at end of fortnight the organisms have disappeared in a large majority of cases unless the cases have
(e) Again sterilise and make
series
across
a
horizontal strokes (0) vertical one without re-
of
A,
Pus and mucus. Vertical stroke. c, Horizontal strokes. B,
moving loop. Sfl Incubate 18-24 hours at 27° C. By this method the portion of specimen most likely to contain B. dysenteriæ is selected. The washing helps to not wanted. The remove various coliform organisms, &c., rubbing and spreading of pus and mucus tends to break up pus cells, liberating bacilli sought for. The stroke method with two sterilisations of loop gives three dilutions, a good separation of colonies on plate, and does away with necessity of using two or more plates for same case sometimes recommended. Selection from Plate
cf Likely Colonies. (a) Discard lactose fermenters. (b) Of the others, pick off with sterilised straight platinum wire one of each type of colony present. Care must be taken to touch only one colony at a time. It is impossible to distinguish, either by naked eye or by means of a lens, between B. dysenteriæ and many other non-lactose fermenters, especially typhoid group, some forms of B. fcccatis alkaligenes, Morgan No. 1, and paracolon bacilli, with any degree of certainty. It should be remembered that colonies of B. dysenteriæ are not clear, as sometimes stated, but are dull grey in colour and slightly opaque. B. Shiga colonies are always more
relapsed. (b) Appearance of specimen.-1. Naked eye:The typical stool is almost completely composed of pus and mucus, in most cases streaked with blood; usually pinkish in colour, giving the impression of greyish-white opaque flakes of In these cases the pus in mucus tinged with blood. collection is simple. When the stool is of a mixed character the pus and mucus portion must be selected for investigation. The sample should be free from urine. The bed-pans must not be washed out with disinfectant before than B. Flexner or Y. being used for obtaining specimen. In some cases of opaque It is interesting to note here that B. dysenteriæ are someFlexner-Y infection the stool is fluid, containing flakes of mucus and pus. As duration of disease increases character times retarded in growth and appear on plate after standing of stool changes. Instead of blood-stained pus and mucus at 300 C. for 24 hours. We have isolated on an average an additional 20 per cent. of B. d ysenterice by incubating in usual one finds btle-stained pus and mucus, especially in Shiga way for 24 hours and then allowing plate to stand another infections. 2. Microscopic Every specimen must be examined 24 hours at 300 C. microscopically for protozoa. Microscopical examination is Identification of Organisms. most important as a guide to selection of stools for culture. This is shown by their effect on sugars, indol, and litmus The appearance in an acute case is almost diagnostic in sera. itself. The field is covered by numbers of cells of various milk, and by agglutination with specific kinds-pus cells chiefly, red blood corpuscles, and epi(a) Subculture colonies selected in Durham’s fermentation thelial cells, especially macrophages. Pus cells and macro- tubes containing litmus-laotose-peptone-water. phages are the important guides. If the sample is non(b) Incubate 10-12 hours at 37° C. cellular, although mucus be present, it is of little use (c) Again discard lactose fermenters. (Note.-It is possible culturing it except in case of possible carrier. We have to pick off lactose fermenters from the plates, as some may cultured many hundreds of stools of this nature with be quite pale when young, and the presence of B. fæcalis alkaligenes sometimes neutralises the acid produced.) negative results.
.
ampoules
from the
same
box
were
used
on
the
surgical side without success. The failures occurred in groups of 3 and 4, and we believe that most, if not all of them, were due to defective solutions. A few drops of Partial failure occurred in 15 cases. chloroform was all that was necessary to finish the operation.
TWO NEW FRENCH METHODS FOR STAINING BLOOD FILMS AND BLOOD PARASITES.
BY L. TRIBONDEAU. We have therefore had about 1’28 per cent. complete MEDICAL OFFICER OF THE FRENCH NAVY. failures and 0 90 per cent. partial failures. There were no deaths from stovaine. THESE two methods, now brought before the readers of Course and extent of ttzv analgesia.-The course of the THE LAN.CET. are very practical, not only because the analgesia varied with amount injected and place of in- technique of the colouration is simple, but especially because after the sensation injection perineal jection. Immediately the staining solutions can be prepared, without difficulty by In about 3-5 minutes (in rare cases 8-10) was abolished. anybody. They are related to the well-known methods of the analgesia would have gradually extended up to the Leishman and provide efficient substitutes for the secret umbilical region if injection was made in fourth Illmbar space of Giemsa and other German writers. and to the ensiform cartilage if in the third. In a few cases processes I. Colouration Method with Stain III. sensibility to heat was retained for some time after complete Of the two methods, this one (neutral solution of eosinate analgesia had been established. Accompanying symptoms.-In a small proportion of cases of methylene-blue converted by ammonia) is more rapid in (less than 2 per cent.) nausea occurred. Actual. vomiting execution and gives more complete results, but it requires occurred in about 3 per cent., bnt never lasted more than a very pure and neutral distilled water. Certain commercial minute. Relaxation of sphincter during analgesia with incon- distilled waters do not possess these qualities.2 Preparation of Stain III.-Heat some distilled water to tinence of faeces occurred in a few cases. Signs of depression, such as yawning, pallor of face, and perspiration, rarely boiling point. Divide. (A)-50 c.cm. in enamelled basin ; add 0-20 g. pure medicinal methyoccurred and have seldom caused anxiety. Pulse at end of blue ; shake to dissolve. operation was in many cases stronger than at beginning. (B)-75 c.cm. in a glass; add 0.30 g. water-soluble postn (French shake to dissolve. Post-operative symptoms.-In about 1 per cent. of the cases Pour B into A by successive fractions. After each addition a certain amount of headache, easily amenable to treatment, of B mix for some time with glass rod ; then place on glass was complained of. The temperature rose a degree or slide drop of mixture from end of rod. This drop is at first two on second and third days in some of onr early cases. i deep blue and free from precipitate. Then, as proportion of Vomiting after operation was conspicuous by its entire eosin is increased, the blue becomes more pale and a preabsence. Paralytic sequences, whether transitory or per- ’, cipitate appears. Finally precipitate increases and colour of have never occurred. manent, liquid turns from blue to rose. Stop addition of eosin as soon as this change occurs. It generally requires a little The Solution Used. more than 50 c.cm. of B to obtain this result, which is quite Stovaine billon was used in about half the cases. Each easy to observe. Add 4 of ammonia to mixture thus obtained. Mix. ampoule contained a little over 2 c.cm. of the following: Heat toc.cm. 120° C. ’in autoclave for 20 minutes in enamelled Borate of epirenin, 000013; stovaine, 0-04; sod. chlor., basin covered with inverted glass funnel. Remove from 0 -0022 ; distilled water to 1-00. autoclave ; stir with rod and allow to cool completely. The amount injected is the same for a big or small operaFilter through small white filter-paper well folded all tion-viz., 2 c.cm. of the solution containing 0-08. In some contents of basin. Discard filtrate. only precipitate, which is almost entirely retained on filter, and of which a cases we injected half or three-quarters of the dose, but the effect was not quite so good and passed off more quickly. small portion remains deposited on side of basin. Dry preAs an exception we may mention colporrhaphies, in which cipitate by placing in incubator at 37° C. the filter widely on several layers of absorbent paper, and also basin. half a dose is sufficient,’bnt even there it is better to inject open When drying is complete (no trace of water must remain a full dose, as it may be found necessary to do something in order that ammonia may be completely volatilised) place more. filter in basin and dissolve as much as possible of dried stain We are now using with even better results a solution by pouring into basin, in successive fractions, 100 c.cm. of (absolute ethyl alcohol, 90 c.cm.; neutral prepared in the school chemical laboratories under the glycerinated alcohol 10 c.cm.) and crushing powder with large glass rod. supervision of Dr. Bahry Bey. The formula used is: glycerine Sod. chlor., 0 °0011 ; stovaine, 0 04 ; water, 1 c.cm. It is Transfer the 100 c.cm. of solution into flask, taking care to transfer also all undissolved stain. Shake flask from time prepared as follows :to time. Filter after 12 to 24 hours. The sodium chloride is dissolved in the water. The soluTechnique of staining.-First fix preparation with undiluted tion is sterilised in the autoclave for 15 minutes and then Stain III. For this, slide carrying blood (previously spread left to cool down. The stovaine is dissolved in solution and in thin film, dried simply by moving in air, and marked off filtered through a good filter previously sterilised. It is by a glass pencil line) is placed on table, film upwards. then heated on a water-bath for ten minutes at 80°-90°C. Cover film with 0’2 c.cm. of Stain III. (approximately After cooling it is stored in sterilised ampoules. The whole 12 drops). Cover with half of Petri dish to prevent too great Allow to act three process is carried on aseptically. Attempts to sterilise the evaporation, especially in summer. stovaine solution by boiling have resulted in the decomposi- i minutes. Then stain by adding to Stain III., on slide itself, 0’6 c.cm. tion of the stovaine and is not necessary, stovaine itself being i ’ of distilled water (approximately 12 drops). Mix water and antiseptic. stain by few movements of slide. Replace on table. Allow Conclusions. to act for average time of 12 minutes without moving. Wash Stovaine analgesia was greatly welcomed by our patients. rapidly with jet of distilled water. Dry immediately (by women have a Most Egyptian great dislike to general anves- passing wet preparation for two seconds over flame and thesia. Many instances have occurred of patients refusing blowing vigorously on it). to have an operation done under chloroform, but agreeing to II. Colouration Method with Stains I. and II. undergo the operation under stovaine analgesia. When good distilled water is not available this second The preparations are made and the stovaine injected before the students come into the theatre. The patient is covered method (neutral solution of eosinate of natural methylene blue alkaline solution of eosinate of methylene blue converted by up with sterilised towels, and is less exposed than during an and ammonia) is preferable to first; it is less delicate. examination in the out-patient department. After having watched this number of patients during their Preparation of Stain L-Pour into heat-resisting flaskNeutral glycerine 5 c.cm. operations and convalescence, and having followed many of 45 c.cm. Absolute ethyl alcohol them for years afterwards, we have no hesitation in asserting Pure medicinal methylene blue............ 0*20 g. that stovaine is, in our opinion, far superior to chloroform in Water soluble eosin (French) 0’05g. all gynaecological operations. During the operation the Dissolve rapidly by plunging flask in very hot-water bath complete relaxation of abdominal muscles and the evenness! and shaking. Allow to cool. Pour into glass-graduated of the analgesia are a great help to the operator. The 1 C. R. Soc. de Biologie de Paris, May-June, 1918. absence of vomiting during the convalescence prevents any One can correct a defective water by redistilling, after addition of strain on the stitches. The freedom from shock and all a 2little silver carbonate obtained by precipitation of a silver nitrate solution with carbonate of soda, but this is complicated. respiratory troubles are points in favour of stovaine.
lene eosin);
’
even
Keep
.....................
..................
............
143 measure; make up to 50 c.cm. with absolute ethyl alcohol. Filter and cork in a flask. Preparation of Stain II.-Pour into heat-resisting flaskNeutral glycerine Ethyl alcohol at 95 per cent............. Pure medicinal methylene blue Water-soluble eosin (French)
.....................
............ ............
25 c.cm. 15 c.cm. 0’20 g.
0’05g.
(c) Age ot’ specimen.-The stool must be cultured in a fresh state, as the bacilli of dysentery ouickly die off in a stool_ If the sample cannot be obtained at a convenient time it should be placed in an ice-chest. A fresh specimen from a case on second day of disease was examined and B. dysenteriæ Shiga was isolated in almost pure culture. Another specimen from the same case was examined as follows :Fresh specimen (fifth day of disease) plated immediately, 27 colonies
Dissolve in water bath as with I. Allow to cool. Pour into glass-graduated measure, and make up to 40 c.cm. with of B. Shiga. 95 per cent. ethyl alcohol. Return into flask. The specimens plated after standing at room temperature or in iceAdd 4 c.cm. of ammonia. Mix. Heat to 120° C. in autoclave for 20 minutes in open flask. Remove from auto- chest gave the number of colonies of B. Shiga stated:Pour into glassclave and allow to cool somewhat. graduated measure and make up to 50 c.cm. with 95 per cent. ethyl alcohol. Return into flask, which is corked only after a dav or two. Teclzrnique of staining.-First fix preparation with unslide on table, film upwards. diluted Stain 1. For this, Cover film with Stain I. Cover with Petri dish. Allow to A fresh specimen from an acute case was cultured, and B. dysenteriæ act three minutes. Wash with jet of distilled water. Get Flexner was isolated easily, the majority of colonies on the plate being rid of surplus water by shaking; wipe under side of slide; of this organism. place it on edge of glass or crystallising dish without The day after a six hours’ old sample from the same case, with film. similar naked-eye and microscopical appearances, was cultured. Not Stain by pouring on preparation Stain II. diluted and hot. one colony of B. dysenteriæ was present on the plate. For this have small test-tube of 1 em. diameter marked at These are most striking facts which prove the absolute 2 c.cm. ; pour distilled water to mark; heat tube held aslant of culturmg fresh specimens. We have recorded in flame until appearance of first bubbles of air ; add to the necessity similar examples. 2 c.cm. of hot water 4 to 5 drops of Stain II. ; pour hot over many It is recommended that the MacConkey’s medium used be film. Allow to act 15 minutes. Wash with jet of distilled water. Dry immediately (heating I made up with double the strength ofneutral red-i.e., and blowing). Remove excess of blue by pouring on dry 0’5 c.cm. of a 1 per cent. solution to every 100 c.cm. of agar. preparation held aslant watery solution of tannin (1 in 20) lYlethod of Plating. until film becomes rose-coloured. (It is necessary to avoid differentiating moist films because preparations would With sterilised platinum loop pick from freah (a) become spotted with blue. The solution of tannin is pre- specimen of fsecea a small portion consisting of pus and pared by dissolving 1 g. of tannin " a 1’alcool" in 20 c.cm. of mucus. very hot water. One adds a little camphor to prevent growth (b) Wash it well in sterilised normal saline and remove Wash at once with distilled water. of moulds.) Dry it immediately with loop, allowing immediately. excess saline to drain away along N.B.-One can omit I., fixing simply with alcohol, but it is sides of tube. then necessary to stain with II. for 30 minutes. (c) Rub and spread washed pus and mucus well on to surface of MacConkey plate nt position (A) shown in diagram. THE ISOLATION OF DYSENTERY BACILLI (cl) Sterilise loop and make vertical stroke (B) from first inocuFROM THE FÆCES.
place
drying
lation.
BY HENRY
WHITEHEAD, M.D., D.P.H. MANCH., M,B., B.S. LOND.,
TEMPORARY CAPTAIN, R.A.M.C. ; BACTERIOLOGIST TO — GENERAL
HOSPITAL; AND
J. KIRKPATRICK, LANCE-CORPORAL, R.A.M.C.
(T.F.).
OUR object is to describe what we consider the best method of isolating B. dysenteriæ from the fxces. Collection and Seleotion of Fæces for Examination. (a) Duration oj disease.-Specimen must be obtained during and as early as possible in acute stage of illness. After a week isolation of B. dysenteriæ becomes increasingly difficult, and at end of fortnight the organisms have disappeared in a large majority of cases unless the cases have
(e) Again sterilise and make
series
across
a
horizontal strokes (0) vertical one without re-
of
A,
Pus and mucus. Vertical stroke. c, Horizontal strokes. B,
moving loop. Sfl Incubate 18-24 hours at 27° C. By this method the portion of specimen most likely to contain B. dysenteriæ is selected. The washing helps to not wanted. The remove various coliform organisms, &c., rubbing and spreading of pus and mucus tends to break up pus cells, liberating bacilli sought for. The stroke method with two sterilisations of loop gives three dilutions, a good separation of colonies on plate, and does away with necessity of using two or more plates for same case sometimes recommended. Selection from Plate
cf Likely Colonies. (a) Discard lactose fermenters. (b) Of the others, pick off with sterilised straight platinum wire one of each type of colony present. Care must be taken to touch only one colony at a time. It is impossible to distinguish, either by naked eye or by means of a lens, between B. dysenteriæ and many other non-lactose fermenters, especially typhoid group, some forms of B. fcccatis alkaligenes, Morgan No. 1, and paracolon bacilli, with any degree of certainty. It should be remembered that colonies of B. dysenteriæ are not clear, as sometimes stated, but are dull grey in colour and slightly opaque. B. Shiga colonies are always more
relapsed. (b) Appearance of specimen.-1. Naked eye:The typical stool is almost completely composed of pus and mucus, in most cases streaked with blood; usually pinkish in colour, giving the impression of greyish-white opaque flakes of In these cases the pus in mucus tinged with blood. collection is simple. When the stool is of a mixed character the pus and mucus portion must be selected for investigation. The sample should be free from urine. The bed-pans must not be washed out with disinfectant before than B. Flexner or Y. being used for obtaining specimen. In some cases of opaque It is interesting to note here that B. dysenteriæ are someFlexner-Y infection the stool is fluid, containing flakes of mucus and pus. As duration of disease increases character times retarded in growth and appear on plate after standing of stool changes. Instead of blood-stained pus and mucus at 300 C. for 24 hours. We have isolated on an average an additional 20 per cent. of B. d ysenterice by incubating in usual one finds btle-stained pus and mucus, especially in Shiga way for 24 hours and then allowing plate to stand another infections. 2. Microscopic Every specimen must be examined 24 hours at 300 C. microscopically for protozoa. Microscopical examination is Identification of Organisms. most important as a guide to selection of stools for culture. This is shown by their effect on sugars, indol, and litmus The appearance in an acute case is almost diagnostic in sera. itself. The field is covered by numbers of cells of various milk, and by agglutination with specific kinds-pus cells chiefly, red blood corpuscles, and epi(a) Subculture colonies selected in Durham’s fermentation thelial cells, especially macrophages. Pus cells and macro- tubes containing litmus-laotose-peptone-water. phages are the important guides. If the sample is non(b) Incubate 10-12 hours at 37° C. cellular, although mucus be present, it is of little use (c) Again discard lactose fermenters. (Note.-It is possible culturing it except in case of possible carrier. We have to pick off lactose fermenters from the plates, as some may cultured many hundreds of stools of this nature with be quite pale when young, and the presence of B. fæcalis alkaligenes sometimes neutralises the acid produced.) negative results.
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