_Circulation_
For the most part, cases showing marked clinical symptoms were studied.
The pulse in severe cases frequently was weak and rapid but regular. In
some cases it was less rapid than the clinical state would seem to
indicate.
_Arterial Blood Pressure_ was low; systolic pressure in severe cases
ranging downward from 95, and diastolic down to 40 or under. In patients
in early stages of convalescence the pressure showed a marked advance
toward normal levels. Arterial blood pressure seemed a reliable general
index of the condition of the patient.
_Venous Blood Pressure._—The observations included patients who a few
hours later expired. The Von Recklinghausen method was used. No marked
abnormality was observed, so other methods of observation were deemed
superfluous.
_Respiration_
In severe cases, frequently it was rapid and of shallow character; but,
like the pulse, often it was less rapid than the clinical state would
seem to indicate.
_Cyanosis_ of dark hue and marked degree was prevalent in the earlier
severe cases, and in some cases appeared entirely out of proportion to
the state of circulation and respiration and to the post-mortem findings
as reported by Dr. Klotz.
_Blood_
Hemorrhage being not uncommon, the blood was tested for coagulability,
but in this respect no marked departure from the normal range was noted.
_Coagulation._—Coagulation time was observed by stirring blood in a test
tube with a wire and noting the time of the appearance of fibrin and by
means of a Biffi-Brooks coagulimeter. The extreme ranges observed were
from 2½ to 5½ minutes. The average by defibrination was 3 minutes and 36
seconds, and by the Biffi-Brooks method 4 minutes and 38 seconds.
_Red Corpuscles._—Osmotic resistance. A number of bloods were examined
by observing their resistance to osmotic laking by exposure to a series
of hypotonic sodium chloride solutions. Though some differences were
observed, from the evidence obtained, it is not permissible to conclude
that such variations were constant or of a significant magnitude.
_Color_ on exposure to air. It was early observed that venous blood from
cyanotic patients was very slow to take on arterial hue on exposure to
air.
_Plasma Bicarbonate._—The plasma bicarbonate was determined in seven
cases by Miss Waddell by the method of Van Slyke and Cullen. In all
except one of these the results were within the normal range as given by
Van Slyke. Three were in the lower normal range, being 54.1, 55.1 and
60.5 respectively, expressed in terms of cubic centimeters of CO_{2}
reduced to 0°, 760 mm. Hg. pressure, bound as bicarbonate by 100 c.cm.
of plasma. Three were in the median range, being 64, 65.5 and 71 c.cm.
In one case the bicarbonate CO_{2} was reduced to 46.6 c.cm.
There seemed to be no constant relation between the apparent severity of
the clinical condition of the patient and the bicarbonate reading. In
the one case in which this was found to be reduced below Van Slyke’s
lower normal limit the blood was taken only a few hours before death.
_Hemoglobin Per Cent._—As determined by the Sahli hemoglobinometer (by
Miss Lee) and as estimated by the total oxygen capacity (Van Slyke
method) (by Dr. Rohde and Mrs. Macklin), the hemoglobin content ranged
within normal levels.
_Relative Volume of Corpuscles._—A limited number of hematokrit tests on
severe cases gave results in normal levels.
_Spectroscopic Studies._—Sera obtained from 20 post-mortem bloods were
examined spectroscopically. In eight an absorption band in the red was
observed. In some instances such a band was observed in blood obtained
shortly after death and before coagulation had occurred, while other
similar bloods, as well as bloods obtained at longer intervals after
death, exhibited no such band. A similar band was observed in one case
from blood obtained from a patient about 12 hours before death from
pneumonia following influenza. Medication was not a causative factor. To
ammonium sulphide the band in the red reacted as methemoglobin and the
position (as estimated by Dr. Menten) corresponded with methemoglobin.
Oxyhemoglobin bands in such bloods occupied normal positions as
determined by Dr. Menten. On diluting such bloods with water no
abnormality in character or position bands was observed, save in one
instance (No. 778 below). This does not, however, disprove the
possibility of such abnormality in the hemoglobin within the cells, for
moderate dilution only of serum rendered the band in the red invisible,
presumably by dilution.
Detailed examination of the absorption bands was made with a direct
reading wave-length Hilger Spectroscope (which was calibrated by
line spectra derived from salts added to an alcohol flame) by Dr.
Menten. This spectroscope had an accuracy of about two Angstroms. In
all, seven post-mortem bloods were examined, viz. autopsy numbers
756, 761, 763, 773, 778, 784, and 787. In five of these, sufficient
serum was obtained to make readings. All gave the two characteristic
oxyhemoglobin bands in the blue-green with centers of the bands at λ
758μμ λ and 542μμ. The second oxyhemoglobin band varied slightly in
width in the different samples. In addition to the two oxyhemoglobin
bands in each of four of the above sera, viz: Nos. 756, 763, 767 and
787, an absorption band in the red was found with the center of the
band as follows: Number 756 at λ 627μμ, number 761 at λ 634μμ,
number 763 at λ 625μμ, and number 787 at λ 634μμ. These bands varied
considerably in intensity and could only be identified when the two
oxyhemoglobin bands were merged and appeared as one broad band. As
controls for the position of the oxyhemoglobin bands two normal
bands were examined, which showed two bands with centers also at λ
758μμ and λ 543μμ. For comparison of the methemoglobin bands of the
above post-mortem bloods, a sample of this hemoglobin compound was
made by adding potassium ferricyanide to normal blood until the
solution became brownish in color. The center of this methemoglobin
band was found at λ 634μμ. In blood from autopsies number 773 and
number 778 sufficient serum could not be obtained to make a reading.
To each of these bloods distilled water was added. The laked blood
of 778 gave a methemoglobin band with the center at λ 632μμ on
examination 24 hours after autopsy. Similar treatment of corpuscles
five days subsequently gave no indication of the presence of any
methemoglobin spectroscopically.
From the serum and from the laked corpuscles of number 784 no trace
of methemoglobin was found when the blood was examined a few hours
after removal at autopsy.
_Oxygen Capacity._—The total oxygen capacity was determined by the Van
Slyke method (by Dr. Rohde and Mrs. Macklin). At this stage the more
pronounced type of influenza had subsided, but in early convalescence
the capacity was within normal ranges.
Other studies using different technique gave concordant results, but
there were indications that oxygen was more slowly absorbed than
normally.
_Oxygen Content of Venous Blood_ measured by the Van Slyke method (by
Dr. Rohde and Mrs. Macklin) on the same bloods examined for total oxygen
capacity seemed to indicate a mild deficiency as compared to normal
bloods.
_Gases, Kinds, Quantity and Rate Yielded to Vacuum._—In general it may
be said that quantitative differences observed are not considered
fundamental, but that the studies indicate abnormal slowness in oxygen
absorption.
_Gases, Quantity and Rate of Absorption on Exposure to Air After
Extraction by Pump._—The results emphasize slowness of oxygen absorption
as compared to normal blood.
The material to be examined was exhausted for three minutes in the
receiver of the Van Slyke apparatus. One c.cm. was then transferred,
with as little exposure to air as possible, to a small empty bottle,
which was then closed and placed in communication with a calibrated,
horizontal tube, containing a segment of alcohol, which served the
dual purpose of a seal and an air volume change indicator. (See Fig.
1.) The apparatus was made in duplicate and mounted on a common
base, so that simultaneous readings on different samples could be
made. After establishing the zero position of the alcohol segment,
the base on which the bottles were mounted was vigorously shaken in
a uniform manner. Ten seconds after the period of shaking, the
volume readings were taken. Successive periods of shaking and
reading were conducted at 30-second intervals, until the test was
completed. Actual volume changes were then calculated, tabulated and
plotted.
The greater confidence is placed on the results obtained by
observing the color of the blood, as described below; but since then
the method has been checked up and the results indicate that the
findings were of sufficient accuracy to warrant their inclusion in
this report.[1]
Footnote 1:
Studies along this line are being made with improved apparatus, the
results of which, together with the description of the apparatus, will
be published elsewhere. (See Am. Gr. Physiol., 1920, li, 195.)
[Illustration: FIG. 1.]
_Effect of Addition of Serum on Behavior on Exposure to Air._—The
persistence of venous hue of blood exposed to air was noted above. It
was observed that the addition of serum from the same blood
conspicuously shortened the time required for such blood to acquire an
arterial hue. The addition of normal serum was more effective in this
respect than pathological serum. Measurements of the rate of absorption
of such blood after the addition of serum indicated acceleration of
oxygen absorption. From this it would seem that the oxygen transmitting
capacity of the serum was diminished.
_Effect of Addition of Dry Sodium Bicarbonate on Behavior on Exposure to
Air._—The addition of a small quantity of dry sodium bicarbonate to a
blood refractory to arterialization on exposure to air enormously
accelerated the process, as judged by the color. To what extent the
change in color may have been due to causes other than oxygen absorption
was not determined.
_Comment_
The most significant positive findings were evidence of deficiency of
serum oxygen transmitting capacity or rate, and the detection in serum
of an absorption band in the red corresponding to methemoglobin. The
presence of the abnormal substance giving rise to the absorption band is
considered of special interest as indicating abnormal chemical
conditions in the blood, rather than material change in hemoglobin
oxygen capacity.
THE BACTERIOLOGY OF EPIDEMIC INFLUENZA WITH A DISCUSSION OF B. INFLUENZÆ
AS THE CAUSE OF THIS AND OTHER INFECTIVE PROCESSES
By W. L. HOLMAN, B. A., M. D.
_Introduction_
In a study of the bacteriology of a respiratory disease such as
influenza, the technical difficulties encountered are very great and
must be overcome before we can draw useful conclusions from the results
obtained or attempt to determine the etiological factors. The important
methods of attacking such a problem include: (1) the study of stained
smears and cultures from the various available materials, along with the
demonstration of the bacteria in the lesions found in the disease by a
study of sections; (2) tests with the various materials to determine the
presence of the causative agent, which includes experiments on man and
animals and is more inclusive than the mere study of the bacteria
isolated; (3) immunological studies of man suffering from the disease,
or of man and animals treated with the materials from the disease; (4)
pathological, clinical and epidemiological studies linked with the
above.
Many of the difficulties and sources of error in these methods are
manifest to all, but certain points may be indicated as more important
in the phases of the work on which I am to report.
_General Methods of Investigation_
Stained smears from the material available. The choice of the material
is of first importance. Sputum to be of any real value must be obtained
from the deeper portions of the respiratory tract, should be as free as
possible from the secretions of the buccal cavity, and should be washed
in saline before it is used. These are considered among the first
requirements in the study of lung infections by the pneumococci and are
equally important in influenza. Swabs from the nasopharynx should be
obtained with the same precautions as are demanded in meningococcal
work. The other available material—such as blood, lung puncture fluid,
pleural fluid and spinal fluid—must be collected with the greatest care.
The staining methods should, naturally, include those which will bring
out the various types of bacteria, and must include the Gram method,
using dilute alcoholic fuchsin (1-20) as the counterstain. The varying
morphology of the B. influenzæ and its frequent minute size make it
difficult to detect. It is not the only Gram negative small bacillus
seen in smears from the throat, but when it occurs in the typical
schools, or where there are numerous bacilli to be seen, its
characteristics are quite definite. I have recently isolated an anærobic
Gram negative bacillus from a series of swabs from the buccal cavity
which suggests in many ways the morphology of the B. influenzæ, which
will indicate one of the many difficulties to be met with in the study
of stained smears. They are, nevertheless, of great use as a control on
cultures, and most helpful in the study of the material from sources
other than the respiratory tract.
Cultures of the bacteria from the various materials. Here we have the
greatest difficulty of all. The medium chosen determines the bacteria
which will appear to predominate, and there is no single medium that
will answer all purposes. Streptococci will appear to be in excess when
serum broth is used, as I have previously shown; pneumococci with
Avery’s pneumococcus medium; and staphylococci, the Gram negative cocci,
and the diphtheria group with Loeffler’s serum. Ordinary blood agar is
perhaps the best general medium for direct and secondary plating. There
have been many special media devised for growing the B. influenzæ, but
the one I have used most and found particularly helpful is heated blood
agar made after the general method of Voges.
The extremely tiny colony of B. influenzæ on ordinary blood agar makes
it particularly difficult to detect, and one is apt to get the wrong
impression of its numbers from the macroscopic appearance of the plate.
In attempts at isolation there must be a liberal use of media in picking
colonies, as many suspicious ones will turn out to be immature growths
of B. xerosis, M. pharyngis (or M. catarrhalis), streptococci, or more
rarely pneumococci and other organisms. Replating from such picks is
frequently necessary, and further plates, from the original culture on
heated blood agar, must often be made before the B. influenzæ can be
isolated. The care required in all stages of the isolation of this
organism, the unstinted use of media for plating and for picks, the
number of stained smears to be studied, and the further transfers
necessary to verify results, all these limit the amount of material
which can be studied with any degree of accuracy. If further the
streptococci, the pneumococci, the Gram negative cocci, the capsulated
Gram negative bacilli and many others are to receive any attention, it
can readily be appreciated that a few cases carefully studied are of far
more value than a large number hurriedly examined in an uncertain
routine.
The pathological study of the same cases on which I have done the
bacteriology will be found in Dr. Klotz’s paper in these communications,
and I will merely refer to some of the bacterial findings in the
sections of the lungs and bronchi. The more inclusive methods which have
been used in attempts to determine the etiological factor in influenza
we have been unable to attempt, but I will refer later in this paper to
the findings of the investigations of others. Immunological studies have
been limited to a few investigations on the presence of agglutinins,
complement binding substance, skin reactions and the amount of
complement present in the sera of certain patients. The epidemiological
and clinical studies are reported by Drs. Johnston and Lichty in this
series of reports.
_Material Studied_
The material used in the study I am reporting included swabs from the
large bronchi and fluid from the lungs and pleural cavities of 32
autopsies, as well as blood cultures from 22 patients and swabs from the
nasopharynx of 31 individuals. Fifteen sera were tested for fixation of
complement with an antigen made from several strains of B. influenzæ.
Fourteen other sera were tested for agglutinins. Complement content was
determined in the sera of 25 patients. Skin tests after the Von Pirquet
method were done on 14 convalescents, and carefully stained
nasopharyngeal smears without cultures were studied from 48 patients.
The chief attention was given to the study of the autopsy material and
we concentrated on the isolation of B. influenzæ. At the same time we
did not neglect the other bacteria making up the flora of the bronchi,
lungs and pleural cavity in these cases. The various types were isolated
and most of them fully identified.
_Technique_
Direct smears were made on sterile slides of all material studied and
stained by Gram’s method. The counterstain was always alcoholic fuchsin
diluted 1-20 in distilled water. Direct cultures were made on a human
blood agar plate containing 5 per cent. blood, which was further smeared
just before use with defibrinated blood. This latter procedure was later
discarded, as it did not appear to assist to any marked extent the
growth of B. influenzæ. Blood broth containing a few drops of
defibrinated blood and blood agar slants smeared with blood were also
used. Heated blood agar (2-3 c.cm. of defibrinated human blood added to
100 c.cm. of ordinary agar at a temperature of from 90 to 100° C., or as
the agar comes from the sterilizer) was used in the last nine cases to
replace the blood agar slant in the direct cultures and as the medium of
choice for transfers of the B. influenzæ.
I prefer the ordinary blood agar plate to the heated blood plate because
the former gives readings which are very helpful in distinguishing
colonies of various types. B. influenzæ appears as clear, tiny,
pinpoint, inert colonies. B. xerosis or the pseudodiphtheria group gives
more opaque but often rather similar colonies. Gram negative cocci as M.
pharyngis siccus have dry, raised, soon becoming wrinkled, inert
colonies, varying greatly in size; M. catarrhalis, more moist, inert
colonies. The cocci of the streptococcus viridans group appear as very
small colonies with greening, or are not infrequently inert, while thin,
flattened colonies with central thickening may sometimes be noted. Those
of the streptococcus hemolyticus group occur as small, frequently
nipple-like colonies with clear, wide zones of hemolysis; pneumococci as
moderately small, moist, dewdrop-like colonies with center collapsing
early and with greening; streptococcus or pneumococcus mucosus as
larger, watery, sticky colonies with greening and frequently an early
clearing near the colonies.