Br.—Bronchus.
Phago.—phagocytosis.
Ht.-Heated blood agar.
B. W.—B. welchii.
Staphylococci develop opaque, paint-like colonies of varying size, with
or without hemolysis, and so do other less frequently found bacteria
give more or less distinctive colonies. The heated blood agar does not
show these differences.
The colonies most liable to be confused with those of B. influenzæ are,
therefore, those of B. xerosis, immature colonies of the Gram negative
cocci and certain colonies of the streptococcus viridans group.
Transfers should always be made to heated blood agar of all colonies
suggestive of B. influenzæ, or when the growth of the B. influenzæ has
only occurred in the more crowded portions of the plate, and it is
difficult to pick pure cultures, attempted pickings should be made to
this medium for further platings. It is frequently necessary to make
further blood agar plates from the original blood agar, blood broth or
heated blood agar cultures after longer incubation periods, depending on
the findings in smears from these media. The heated blood agar is the
best of these to encourage the growth of B. influenzæ. It must, however,
be used at once, or within a very few days of its preparation, and
cannot be kept on hand as a stock medium. I have not found it as useful
for plating because of the difficulty of differentiating colonies. The
phenomenon of the star-like and more luxuriant growth of the colonies of
B. influenzæ about colonies of other bacteria has often been noted, and
will be referred to in a later portion of this report. Here it may be
said that this is at times a marked feature of certain mixtures and must
be recognized in studying the plates. The finding of B. influenzæ in
picks from apparently isolated colonies of other forms is not uncommon,
and is the same type of difficulty which I have discussed in papers on
streptococci. It is important to recall, in connection with cultures
taken from the lungs at autopsy, the experimental work of Norris and
Pappenheimer, who showed that B. prodigiosus put in the mouth
immediately after death could be recovered from the lungs in over 50 per
cent. of the cases studied.
_Results of the Author_
In Table I are shown my results from the 32 cases which came to autopsy.
The B. influenzæ was isolated from one or more sources in 25, making a
total of 78 per cent. Most of the negative cases probably also had this
organism, but I did not grow it from the material which I used for
culturing. The work of others would indicate that it may have been
present in other regions, such as the sinuses of the head or other
portions of the lung and respiratory tract. The positive results show B.
influenzæ present in 20 out of 30 cases from the bronchi; in 13 of 28
from the lungs; in 2 of 14 from the pleural cavity; in 9 of 26 from both
bronchi and lung where both were cultured; in 8 of 26 from the bronchi
with the lung negative; in 3 of 26 from the lung with the bronchi
negative; once of 10 from the pleural cavity with both the bronchi and
the lung negative, and once from all three sources.
The negative results occurred in seven cases. In three of these (749,
750, 756) B. coli overgrew the cultures from the bronchus, in two also
from the lung, and in one, without lung culture, from bronchus and
pleural cavity. The mere presence of B. coli, however, did not preclude
the isolation of B. influenzæ, as is seen in cases 746, 758, 761 and
765. The finding of B. coli would suggest a post-mortem invasion. The
hours after death before the autopsy was done were in these seven cases,
½, 15, 6, 18, 16, 19, 16, respectively. That delay in performing the
autopsy, as emphasized by Spooner, Scott and Heath, adds to the
difficulty is self-evident, but successful isolations of B. influenzæ
have been obtained after even longer periods than in the negative cases
(761). In the fourth negative case (763) the bronchus was not cultured.
A pneumococcus was grown from the pleural cavity and no growth was
obtained from the lung. In the original culture from the pleural cavity
influenza-like forms were seen but could not be isolated. In the fifth
case (767) a blood culture three days before death gave a growth of
pneumococcus mucosus which was also grown from the lung at autopsy.
Direct smear from the bronchus showed very few influenza-like forms. Our
sixth negative finding was in a case of 20 days’ illness, the patient
having had a recurrence (773). Staphylococcus pyogenes aureus,
streptococcus viridans and B. xerosis were grown from the bronchus. Only
a sarcina form grew from the lung, and a further probable air
contamination occurred on the media from the cultures of the pleural
cavity. The B. xerosis colonies were confusing, picked as possible B.
influenzæ, and, before this was discovered, the overgrowth prevented
further attempts to isolate the influenza bacilli. The last unsuccessful
case was one with a general infection of a hemolytic streptococcus from
an acute otitis media. The streptococcus was isolated from the bronchus,
lung, spleen, arm vein and the middle ear at autopsy.
It will be seen that in these seven negative cases technical
difficulties prevented the isolation of the B. influenzæ, even if it had
been present. I would not, therefore, conclude that the organisms were
necessarily absent, but rather that we have failed either to secure
material from the focus of infection or on account of the other reasons
mentioned.
It is very evident that a variety of secondary organisms very frequently
overgrow the field and become numerically predominant. In our first case
staphylococcus pyogenes aureus overgrew all the other organisms present
in cultures from the lung material. B. influenzæ was, however, seen in
the original 24-hour blood agar culture. It required 9 blood agar plates
before the organism could be isolated. In another case 10 plates were
used for the isolation.
The findings of the bacteria in the lung sections are particularly
interesting and instructive. The entire series of cases have not been
completely studied, so I am unable to tabulate the findings. In cases
761 and 762 sections of the lung showed influenza-like bacilli to be
almost pure in the earlier stages of the process, while in areas with
purulent foci pneumococcus-like and other Gram positive cocci were also
numerous. In some cases B. influenzæ-like organisms were to be seen in
overwhelming numbers. In others they were scarce, while in some nothing
resembling B. influenzæ could be found in the sections. Positive
cultures were often independent of whether the influenza-like forms were
to be seen in smears or sections or not, although they were found in the
great majority of the cases. The findings in the direct smears and the
bacteriological results make useful material for comparison.
Swabs from the nasopharynx were cultured from 31 individuals; nearly all
of these were cases suspected of diphtheria or as carrying the
diphtheria bacillus, and no particular effort was made to isolate the B.
influenzæ. They were seen in the mixed culture occasionally. In the last
eight cases the heated blood agar, ordinary blood agar and Loeffler’s
serum were seeded from the throat swabs. B. influenzæ practically
overgrew all the other bacteria from seven of these cases on the heated
blood agar medium and was isolated without difficulty; all eight showed
M. catarrhalis. The two other media gave little or no evidence of the
presence of B. influenzæ. As I have said above, our attention was
concentrated on the autopsy material. These cultures from the throat
were simply made to demonstrate the usefulness of the heated blood agar.