The general characteristics of toxins have been described (Chapter
XII). It has been stated that they are more or less specific in
their action on cells. In order to affect a cell it is evident that
a toxin must enter into chemical combination with it. This implies
that the toxin molecule possesses a chemical group which can combine
with a receptor of the cell. This group is called the _haptophore_ or
combining group. The toxic or injurious portion of the toxin molecule
is likewise spoken of as the _toxophore_ group. When a toxin is
introduced into the body its _haptophore_ group combines with suitable
_receptors_ in different cells of the body. If not too much of the
toxin is given, instead of injuring, it acts as a chemical stimulus
to the cell in the manner already described. The cell in response
produces more of the specific thing, which in this instance is more
receptors which can combine with the toxin, _i.e._, with its haptophore
group. If the stimulus is kept up, more and more of these receptors
are produced until an excess for the cell accumulates, which excess is
excreted from the individual cell and becomes free in the blood. These
free receptors have, of course, the capacity to combine with toxin
through its haptophore group. When the toxin is combined with these
free receptors, it cannot combine with any other receptors, _e.g._,
those in another cell and hence cannot injure another cell. These free
receptors constitute, in this case, _antitoxin_, so-called because
they can combine with toxin and hence neutralize it. Antitoxins are
specific--that is, an antitoxin which will combine with the toxin of
_Clostridium tetani_ will not combine with that of _Corynebacterium
diphtheriæ_ or of _Clostridium botulinum_, or of any other toxin,
vegetable or animal.
When a toxin is kept in solution for some time or when it is heated
above a certain temperature (different for each toxin) it loses its
poisonous character. It may be shown, however, that it is still capable
of uniting with antitoxin, and preventing the latter from uniting with
a fresh toxin. This confirms the hypothesis that a toxin molecule has
at least two groups: a combining or _haptophore_, and a poisoning or
_toxophore_ group. A toxin which has lost its poisonous property, its
toxophore group, is spoken of as a _toxoid_. The theory of antitoxin
formation is further supported by the fact that the proper introduction
of _toxoid_, the _haptophore_ group, and hence the real stimulus, can
cause the production of _antitoxin_ to a certain extent at least.
The close relationship between toxins and enzymes has already been
pointed out. This is still further illustrated by the fact that when
enzymes are properly introduced into the tissues of an animal there
is formed in the animal an _antienzyme_ specific for the enzyme in
question which can prevent its action. The structure of enzymes,
as composed of a _haptophore_, or uniting, and a _zymophore_ or
_digesting_ (or other activity) group, is similar to that of toxins,
and _enzymoids_ or enzymes which can combine with the substance acted
on but not affect it further, have been demonstrated.
These free cell receptors, antitoxins or antienzymes, which are
produced in the body by the proper introduction of toxins or enzymes,
respectively, have the function of _combining_ with these bodies
_but no other action_. As was pointed out above, this is sufficient
to neutralize the toxin or enzyme and prevent any injurious effect
since they can unite with nothing else. Since these receptors are the
simplest type which has been studied as yet, they are spoken of by
Ehrlich as _receptors of the first order_. Other antibodies which are
likewise free receptors of the first, order and have the function of
combining only have been prepared and will be referred to in their
proper connection. They are mainly of theoretical interest.
Ehrlich did a large part of his work on toxins and antitoxins
with _ricin_, the toxin of the castor-oil bean, _abrin_, from the
jequirity bean, _robin_ from the locust tree, and with the toxins and
antitoxins for diphtheria and tetanus. Antitoxins have been prepared
experimentally for a large number of both animal and vegetable poisons,
including a number for bacterial toxins. The only ones which, as yet,
are of much practical importance are _antivenin_ for snake poison, (not
a true toxin, however, see p. 275), _antipollenin_ (supposed to be
for the toxin of hay fever) and the antitoxins for the true bacterial
toxins of _Corynebacterium diphtheriæ_ and _Clostridium tetani_.
The method of preparing antitoxins is essentially the same in all
cases, though differing in minor details. For commercial purposes
large animals are selected, usually horses, so that the yield of
serum may be large. The animals must, of course, be vigorous, free
from all infectious disease. The first injection given is either a
relatively small amount of a solution of toxin or of a mixture of
toxin and antitoxin. The animal shows more or less reaction, increased
temperature, pulse and respiration and frequently an edema at the
point of injection, unless this is made intravenously. After several
days to a week or more, when the animal has recovered from the first
injection, a second stronger dose is given, usually with less reaction.
Increasingly large doses are given at proper intervals until the animal
may take several hundred times the amount which would have been fatal
if given at first. The process of immunizing a horse for diphtheria or
tetanus toxin usually takes several months. Variations in time and in
yield of antitoxin are individual and not predictable in any given case.
After several injections a few hundred cubic centimeters of blood
are withdrawn from the jugular vein and serum from this is tested
for the amount of antitoxin it contains. When the amount is found
sufficiently large (250 “units” at least for diphtheria per cc.)[24]
then the maximum amount of blood is collected from the jugular with
sterile trocar and cannula. The serum from this blood with the addition
of an antiseptic (0.5 per cent. phenol, tricresol, etc.) constitutes
“antidiphtheritic serum” or “antitetanic serum,” etc. All sera which
are put on the market must conform to definite standards of strength
expressed in “units” as determined by the U. S. Hygienic Laboratory.
In reality a “unit” of diphtheria antitoxin in the United States is
an amount equivalent to 1 cc. of a given solution of a _standard_
diphtheria _antitoxin_ which is kept at the above-mentioned laboratory.
This statement, of course, gives no definite idea as to the amount
of antitoxin actually in a “unit.” Specifically stated, a “unit” of
antitoxin contains approximately the amount which would protect a 250
gram guinea-pig from 100 minimum lethal doses of diphtheria toxin, or
protect 100 guinea-pigs weighing 250 grams each from one minimum lethal
dose each. The minimum lethal dose (M. L. D.) of diphtheria toxin is
the least amount that will kill a guinea-pig of the size mentioned
within four days. Since toxins on standing change into toxoids to a
great extent, the amount, of antitoxin in a “unit,” though protecting
against 100 M. L. D., in reality would protect against about 200 M. L.
D. of toxin containing no toxoid.
The official unit for tetanus antitoxin is somewhat different, since it
is standardized against a _standard toxin_ which is likewise kept at
the Hygienic Laboratory. The unit is defined as “ten times the amount
of antitoxin necessary to protect a 350 g. guinea-pig for 96 hours
against the _standard test dose_” of the standard toxin. The standard
test dose is 100 M. L. D. of toxin for a 350 g. guinea-pig. To express
it another way, one could say that a “unit” of tetanus antitoxin
would protect one thousand 350 g. guinea-pigs from 1 M. L. D. each of
standard tetanus toxin.
Various methods have been devised for increasing the amount of
antitoxin in 1 cc. of solution by precipitating out portions of the
blood-serum proteins and at the same time concentrating the antitoxin
in smaller volume. It is not considered necessary in a work of this
character to enter into these details nor to discuss the process of
standardizing antitoxin so that the exact amount of “units” per cc. may
be known.
CHAPTER XXVIII.
RECEPTORS OF THE SECOND ORDER.
AGGLUTININS.
Charrin and Rogers appear to have been the first (1889) to observe the
clumping together of bacteria (_Pseudomonas pyocyanea_) when mixed
with the blood serum of an animal immunized against them. Gruber and
Durham (1896) first used the term “agglutination” in this connection
and called the substance in the blood-serum “agglutinin.” Widal (1896)
showed the importance of the reaction for diagnosis by testing the
blood serum of an infected person against a known culture (typhoid
fever).
It is now a well-known phenomenon that the proper injection of cells
of any kind foreign to a given animal will lead to the accumulation in
the animal’s blood of substances which will cause a clumping together
of the cells used when suspended in a suitable liquid. The cells settle
out of such suspension much more rapidly than they would otherwise
do. This clumping is spoken of as “agglutination” and the substances
produced in the animal are called “agglutinins.” If blood cells are
injected then “hemagglutinins” result: if bacterial cells “bacterial
agglutinins” for the particular organism used as “glanders agglutinin”
for _Pfeifferella mallei_, “abortion agglutinin” for _Bacterium
abortus_, “typhoid agglutinin” for _Bacterium typhosum_, etc.
The phenomenon may be observed either under the microscope or in small
test-tubes, that is, either _microscopically_ or _macroscopically_.
In this case the cells introduced, or more properly, some substances
within the cells, act as stimuli to the body cells of the animal
injected to cause them to produce more of the specific cell receptors
which respond to the stimulus. The substance within the introduced
cell which acts as a stimulus (_antigen_) to the body cells is called
an “_agglutinogen_.” That “agglutinogen” is present in the cell has
been shown by injecting animals experimentally with extracts of cells
(bacterial and other cells) and the blood serum of the animal injected
showed the presence of agglutinin for the given cell. It will be
noticed that the receptors which become the free agglutinins have at
least _two functions_, hence at least _two chemical groups_. They must
combine with the foreign cells and also bring about their clumping
together, their agglutination. Hence it can be stated technically that
an agglutinin possesses a _haptophore group_ and an _agglutinating
group_.
It is probable that the agglutination, the clumping, is a secondary
phenomenon depending on the presence of certain salts and that the
agglutinin acts on its antigen as an enzyme, possibly a “splitting”
enzyme. This is analogous to what occurs in the curdling of milk
by rennet and in the coagulation of blood. This probability is
substantiated by the fact that suspensions of bacteria may be
“agglutinated” by appropriate strengths of various acids.
The formation of agglutinin in the body for different bacteria does
not as yet appear to be of any special significance in protecting the
animal from the organism, since the bacteria are not killed, even
though they are rendered non-motile, if of the class provided with
flagella, and are clumped together. The fact that such bodies are
formed, however, is of decided value in the diagnosis of disease, and
also in the identification of unknown bacteria.
In many bacterial diseases, agglutinins for the particular organism
are present in the blood serum of the affected animal. Consequently
if the blood serum of the animal be mixed with a suspension of the
organism supposed to be the cause of the disease and the latter be
agglutinated, one is justified in considering it the causative agent,
provided certain necessary conditions are fulfilled. In the first place
it must be remembered that the blood of normal animals frequently
contains agglutinins (“normal agglutinins”) for many different
bacteria when mixed with them in full strength. Hence the serum must
always be diluted with physiological salt solution (0.85 per cent.).
Further, closely related bacteria may be agglutinated to some extent by
the same serum. It is evident that if they are closely related, their
protoplasm must contain some substances of the same kind to account for
this relationship. Since some of these substances may be agglutinogens,
their introduction into the animal body will give rise to agglutinins
for the related cells, as well as for the cell introduced. The
agglutinins for the cell introduced “chief agglutinins,” will be
formed in larger quantity, since a given bacterial cell must contain
more of its own agglutinogen than that of any other cell. By _diluting
the blood serum_ from the animal to be tested the agglutinins for the
related organisms (so-called “coagglutinins” or “partial agglutinins”)
will become so much diminished as to show no action, while the
agglutinin for the specific organism is still present in an amount
sufficient to cause its clumping. _Agglutinins are specific for their
particular agglutinogens_, but since a given blood serum may contain
many agglutinins, the _serum’s specificity for a given bacterium_
can be determined only by diluting it until this bacterium alone
is agglutinated. Hence the necessity of diluting the unknown serum
in varying amounts when testing against several known bacteria to
determine for which it is specific, _i.e._, which is the cause of the
disease in the animal.
The agglutinins in the serum may be removed from it by treating it with
a suspension of the cells for which agglutinins are present. If the
“chief” cell is used all the agglutinins will be absorbed. If related
cells are used, only the agglutinins for this particular kind are
removed. These “absorption tests” furnish another means of determining
specificity of serum, or rather of determining the “chief agglutinin”
present.
Just as an unidentified _disease_ in an animal may be determined by
testing its serum as above described against _known_ kinds of bacteria,
so _unknown bacteria_ isolated from an animal, from water, etc., may
be identified by testing them against the _blood sera_ of different
animals, each of which has been properly inoculated with a different
kind of _known bacteria_. If the unknown organism is agglutinated
by the blood of one of the animals in high dilution, and not by the
others, evidently the bacterium is the same as that with which the
animal has been inoculated, or _immunized_, as is usually stated. This
method of identifying cultures of bacteria is of wide application,
but is used practically only in those cases where other methods of
identification are not readily applied, and especially where other
methods are _not sufficient_ as in the “intestinal group” of organisms
in human practice.
The diagnosis of disease in an animal by testing its serum is also a
valuable and much used procedure. This is the method of the “Widal” or
“Gruber-Widal” test for typhoid fever in man and is used in veterinary
practice in testing for glanders, contagious abortion, etc. In some
cases a dilution of the serum of from 20 to 50 times is sufficient for
diagnosis (Malta fever), in most cases, however, 50 times is the lowest
limit. Evidently the greater the dilution, that is, the higher the
“titer,” the more specific is the reaction.
PRECIPITINS.
Since agglutinins act on bacteria, probably through the presence of
substances within the bacterial cell, it is reasonable to expect that
if these substances be dissolved out of the cell, there would be some
reaction between their (colloidal) solution and the same serum. As
a matter of fact Kraus (1897) showed that broth cultures freed from
bacteria by porcelain filters do show a precipitate when mixed with
the serum of an animal immunized against the particular bacterium and
that the reaction is specific under proper conditions of dilution.
It was not long after Kraus’s work until the experiments were tried
of “immunizing” an animal not against a bacterium or its filtered
culture, but against (colloidal) solutions of proteins, such as white
of egg, casein of milk, proteins of meat and of blood serum, vegetable
proteins, etc. It was ascertained that in all these cases the animal’s
serum contains a substance which causes a _precipitate_ with solutions
of the protein used for immunization. The number of such precipitating
serums that have been made experimentally is very large and it appears
that protein from any source when properly introduced into the blood
or tissues of an animal will cause the formation of a precipitating
substance for its solutions. This substance is known, technically as a
“_precipitin_.” The protein used as antigen to stimulate its formation,
or some part of the protein molecule (haptophore group), which acts
as stimulus to the cell is spoken of as a “precipitinogen,” both
terms after the analogy of “agglutinin” and “agglutinogen.” In fact
the specific precipitation and agglutination are strictly analogous
phenomena. Precipitins act on proteins in (colloidal) _solution_ and
cause them to settle out, agglutinins act on substances within cells
which cells are in _suspension_ in a fluid and cause the cells to
settle out. Ehrlich’s theory of the formation of precipitins is similar
to that of agglutinins, and need not be repeated. Substitute the
corresponding words in the theory of formation of agglutinins as above
given and the theory applies.
The precipitin reaction has not found much practical use in
bacteriology largely because the “agglutination test” takes its place
as simpler of performance and just as accurate. The reaction is,
however, generally applicable to filtrates of bacterial cultures and
could be used if needed. The so-called “mallease” reaction in glanders
is an instance.
Precipitins find their greatest usefulness in legal medicine and
in food adulteration work. As was noted above, if animals, rabbits
for example, are immunized with the blood of another animal (human
beings) precipitins are developed which are specific for the injected
blood with proper dilution. This forms an extremely valuable means
of determining the _kind of blood_ present in a given spot shown by
chemical and spectroscopic tests to be blood and has been adopted as
a legal test in countries where such rules of procedure are applied.
Similarly the test has been used to identify the different kinds of
meat in sausage, and different kinds of milk in a mixture. An extract
of the sausage is made and tested against the serum of an animal
previously treated with extract of horse meat, or hog meat, or beef,
etc., the specific precipitate occurring with the specific serum. Such
reactions have been obtained where the protein to be tested was diluted
100,000 times and more. Biological relationships and differences have
been detected by the reaction. Human immune serum shows no reaction
with the blood of any animals except to a slight extent with that of
various monkeys, most with the higher, very slight with the lower Old
World and scarcely any with New World monkeys.
It is a fact of theoretical interest mainly that if agglutinins
and precipitins themselves be injected into an animal they will
act as _antigens_ and cause the formation of _antiagglutinins_ or
_antiprecipitins_, which are therefore receptors of the first order
since they simply combine with these immune bodies to neutralize their
action, have only a combining or haptophore group. Also if agglutinins
or precipitins be heated to the proper temperature they may retain
their combining power but cause no agglutination or precipitation,
_i.e._, they are converted into agglutinoid or precipitinoid
respectively after the analogy of toxin and toxoid.
Precipitins like agglutinins possess at least two groups--a combining
or _haptophore_ group and a _precipitating_ (sometimes called
zymophore) group. Hence they are somewhat more complex than antitoxins
or antienzymes which have a combining group only. For this reason
Ehrlich classes agglutinins and precipitins as _receptors of the second
order_.
CHAPTER XXIX.
RECEPTORS OF THE THIRD ORDER.
CYTOLYSINS.
Before Koch definitely proved bacteria capable of causing disease
several physiologists had noted that the red corpuscles of certain
animals were destroyed by the blood of other animals (Creite, 1869,
Landois, 1875), and Traube and Gescheidel had shown that freshly drawn
blood destroys bacteria (1874). It was not until about ten years
afterward that this action of the blood began to be investigated in
connection with the subject of immunity. Von Fodor (1885) showed that
saprophytic bacteria injected into the blood are rapidly destroyed.
Flügge and his pupils, especially Nuttall in combating Metchnikoff’s
theory of phagocytosis, announced in 1883, studied the action of the
blood on bacteria and showed its destructive effect (1885-57). Nuttall
also showed that the blood lost this power if heated to 56°. Buchner
(1889) gave the name “alexin” (from the Greek “to ward off”) to the
destroying substance and showed that the substance was present in
the _blood serum_ as well as in the whole blood, and that when the
serum lost its power to dissolve, this could be restored by adding
fresh blood. Pfeiffer (1894) showed that the destructive power of the
blood of animals immunized against bacteria (cholera and typhoid) was
markedly specific for the bacteria used. He introduced a mixture of
the blood and the bacteria into the abdominal cavity of the immunized
animal or of a normal one of the same species and noted the rapid
solution of the bacteria by withdrawing portions of the peritoneal
fluid and examining them (“Pfeiffer’s phenomenon”). Belfanti and
Carbone and especially Bordet (1898) showed the specific dissolving
action of the serum of one animal on the blood corpuscles of another
animal with which it had been injected. Since this time the phenomenon
has been observed with a great variety of cells other than red blood
corpuscles and bacteria--leukocytes, spermatozoa, cells from liver,
kidney, brain, epithelia, etc., protozoa, and many vegetable cells.
It is therefore a well-established fact that the proper injection of
an animal with almost any cell foreign to it will lead to the blood of
the animal injected acquiring the power to injure or destroy cells of
the same kind as those introduced. The destroying power of the blood
has been variously called its “cytotoxic” or “cytolytic” power, though
the terms are not strictly synonymous since “cytotoxic” means “cell
poisoning” or “injuring,” while “cytolytic” means “cell dissolving.”
The latter term is the one generally used and there is said to be
present in the blood a specific “cytolysin.” The term is a general one
and a given cytolysin is named from the cell which is dissolved, as a
_bacteriolysin_, a _hemolysin_ (red-corpuscle-lysin), _epitheliolysin_,
_nephrolysin_ (for kidney cells), etc. If the cell is _killed_ but
not _dissolved_ the suffix “cidin” or “toxin” is frequently used as
“bacteriocidin,” “spermotoxin,” “neurotoxin,” etc.
The use of the term “cytolysin” is also not strictly correct, though
convenient, for the process is more complex than if _one substance
only_ were employed. As was stated above, the immune serum loses its
power to dissolve the cell if it is heated to 55° to 56° for half an
hour, it is _inactivated_. But if there be added to the heated or
inactivated serum a small amount of _normal serum_ (which contains only
a very little cytolytic substance, so that it has no dissolving power
when so diluted) the mixture again becomes cytolytic. It is evident
then that in cytolysis there are _two distinct substances_ involved,
one which is _present in all serum, normal or immune_, and the other
_present only in the immune cytolytic_ serum. This may be more apparent
if the facts are arranged in the following form:
I. Immune serum dissolves cells in high dilution.
II. Heated immune serum does not dissolve cells.
III. Normal serum in high dilution does not dissolve cells.
II. + III., _i.e._, Heated immune serum plus diluted normal serum
dissolves cells.
Therefore, there is something in heated immune serum necessary for
cell dissolving and something different in diluted normal serum which
is necessary. This latter something is present in unheated immune
serum also, and is destroyed by heat. Experiment has shown that it is
the substance present in all serum both normal and immune that is the
true dissolving body, while the immune substance serves to unite this
body to the cell to be destroyed, _i.e._, to the antigen. Since the
immune body has therefore _two uniting groups_, one for the dissolving
substance and one for the cell to be dissolved, Ehrlich calls it the
“_amboceptor_.” He also uses the word “_complement_” to denote the
dissolving substance, giving the idea that it completes the action of
dissolving after it has been united to the cell by the amboceptor, thus
replacing Buchner’s older term “alexin” for the same dissolving body.
AMBOCEPTORS.
The theory of formation of amboceptors is similar to that for the
formation of the other types of antibodies. The cell introduced
contains some substance, which acts as a chemical stimulus to some of
the body cells provided with proper receptors so that more of these
special receptors are produced, and eventually in excess so that they
become free in the blood and constitute the free amboceptors. It will
be noticed that these free receptors differ from either of the two
kinds already described in that they have _two uniting groups_, one for
the antigen (cell introduced) named _cytophil-haptophore_, the other
for the complement, _complementophil haptophore_. Hence amboceptors
are spoken of as _receptors of the third order_. They have no other
function than that of this double combining power. The action which
results is due to the third body--the complement. It will be readily
seen that complement must possess at least two groups, a combining or
_haptophore group_ which unites with the amboceptor, and an active
group which is usually called the _zymophore_ or _toxophore_ group.
Complements thus resemble either toxins, where the specific cell
(antigen) is injured or killed, or enzymes, in case the cell is
likewise dissolved. This action again shows the close relation between
toxins and enzymes. Complement may lose its active group in the same
way that toxin does and becomes then _complementoid_. Complement is
readily destroyed in blood or serum by heating it to 55° to 56° for
half an hour, and is also destroyed spontaneously when serum stands for
a day or two, less rapidly at low temperature than at room temperature.
Amboceptors appear to be _specific_ in the same sense that agglutinins
are. That is, if a given cell is used to immunize an animal, the
animal’s blood will contain amboceptors for the cell used and also for
others closely related to it. Immunization with spermatozoa or with
epithelial or liver cells gives rise to amboceptors for these cells
and also for red blood corpuscles and other body cells. A typhoid
bactericidal serum has also some dissolving effect on colon bacilli,
etc. Hence a given serum may contain a chief amboceptor and a variety
of “coamboceptors,” or one amboceptor made up of a number of “partial
amboceptors” each specific for its own antigen (“amboceptorogen”).
Amboceptors may combine with other substances than antigen and
complement, as is shown by their union with lecithin and other
“lipoids,” though these substances seem capable of acting as complement
in causing solution of blood corpuscles.
COMPLEMENTS.
As to whether complements are numerous, as Ehrlich claims, or there
is only one complement, according to Buchner and others, need not be
discussed here. In the practical applications given later as means of
diagnosis it is apparent that all the complement or complements are
capable of uniting with at least two kinds of amboceptors.
If complement be injected into an animal it may act as an antigen and
give rise to the formation of _anticomplement_ which may combine with
it and prevent its action and is consequently analogous to antitoxin.
If amboceptors as antigen are injected into an animal there will be
formed by the animal’s cells _antiamboceptors_. As one would expect,
there are two kinds of antiamboceptors, one for each of its combining
groups, since it has been stated that it is always the combining group
of any given antigen that acts as the cell stimulus. Hence we may have
an “anticytophil amboceptor” or an “anticomplementophil amboceptor.”
These antiamboceptors and the anticomplements are analogous to
antitoxin, antiagglutinin, etc., and hence are receptors of the first
order.
ANTISNAKE VENOMS.
A practical use of antiamboceptors is in antisnake venoms. Snake
poisons appear to contain only _amboceptors_ for different cells of the
body. In the most deadly the amboceptor is specific for nerve cells
(cobra), in others for red corpuscles, or for endothelial cells of the
bloodvessels (rattlesnake). The complement is furnished by the blood
of the individual bitten, that is, in a sense the individual poisons
himself, since he furnishes the destroying element. The antisera
contain antiamboceptors which unite with the amboceptor introduced and
prevent it joining to cells and thus binding the complement to the
cells and destroying them. With this exception these antibodies are
chiefly of theoretical interest.
FAILURE OF CYTOLYTIC SERUMS.
The discovery of the possibility of producing a strongly bactericidal
serum in the manner above described aroused the hope that such sera
would prove of great value in passive immunization and serum treatment
of bacterial diseases. Unfortunately such expectations have not been
realized and no serum of this character of much practical importance
has been developed as yet (with the possible exception of Flexner’s
antimeningococcus serum in human practice. What hog cholera serum is
remains to be discovered).
The reasons for the failure of such sera are not entirely clear.
The following are some that have been offered: (1) Amboceptors do
not appear to be present in very large amount so that relatively
large injections of blood are necessary, which is not without risk
in itself. (2) Since the complement is furnished by the blood of the
animal to be treated, there may not be enough of this to unite with a
sufficient quantity of amboceptor to destroy all the bacteria present,
hence the disease is continued by those that escape. (3) Or the
complement may not be of the right kind to unite with the amboceptor
introduced, since this is derived from the blood of a _heterologous_
(“other kind”) species. In hog-cholera serum, if it is bactericidal,
this difficulty is removed by using blood of a _homologous_ (“same
kind”) animal. Hence Ehrlich suggested the use of apes for preparing
bactericidal sera for human beings. The good results which have been
reported in the treatment of human beings with the serum of persons
convalescing from the same disease indicate that this lack of proper
complement for the given amboceptor is probably a chief factor in the
failure of sera from lower animals. (4) The bacteria may be localized
in tissues (lymph glands), within cavities (cranial, peritoneal), in
hollow organs (alimentary tract), etc., so that it is not possible to
get at them with sufficient serum to destroy all. This difficulty is
obviated by injecting directly into the spinal canal when Flexner’s
antimeningococcus serum is used. (5) Even if the bacteria are dissolved
it does not necessarily follow that their _endotoxins_ are destroyed.
These may be merely liberated and add to the danger of the patient,
though this does not appear to be a valid objection. (6) Complement
which is present in such a large excess of amboceptor may just as
well unite with amboceptor which is not united to the bacteria to be
destroyed as with that which is, and hence be actually prevented from
dissolving the bacteria. Therefore it is difficult to standardize the
serum to get a proper amount of amboceptor for the complement present.
COMPLEMENT-FIXATION TEST.
Although little practical use has been made of bactericidal sera,
the discovery of receptors of this class and the peculiar relations
between the antigen, amboceptor and complement have resulted in
developing one of the most delicate and accurate methods for the
diagnosis of disease and for the recognition of small amounts of
specific protein that is in use today.
This method is usually spoken of as the “complement-fixation” or the
“complement-deviation test” (“Wassermann test” in syphilis) and is
applicable in a great variety of microbial diseases, but it is of
practical importance in those diseases only where other methods are
uncertain--syphilis in man, concealed glanders in horses, contagious
abortion in cattle, etc. A better name would be the “Unknown Amboceptor
Test” since it is the amboceptor that is searched for in the test by
making use of its power to “fix” complement.
The principle is the same in all cases. The method depends, as
indicated above, on the ability of complement to combine with at least
two amboceptor-antigen systems, and on the further fact that if the
combination with one amboceptor-antigen system is once formed, it
does not dissociate so as to liberate the complement for union with
the second amboceptor-antigen system. If an animal is infected with a
microörganism and a part of its defensive action consists in destroying
the organisms in its blood or lymph, then it follows from the above
discussion of cytolysins that there will be developed in the blood of
the animal amboceptor specific for the organism in question. If the
presence of this _specific amboceptor_ can be detected, the conclusion
is warranted that the organism for which it is specific is the cause of
the disease. Consequently what is sought in the “complement-fixation
test” is a _specific amboceptor_. In carrying out the test, blood
serum from the suspected animal is collected, heated at 56° for half
an hour to destroy any complement it contains and mixed in definite
proportions with the specific antigen and with complement. The
antigen is an extract of a diseased organ (syphilitic fetal liver,
in syphilis), a suspension of the known bacteria, or an extract of
these bacteria. Complement is usually derived from a guinea-pig,
since the serum of this animal is higher in complement than that
of most animals. The blood of the gray rat contains practically as
much. If the specific amboceptor is present, that is, if the animal
is infected with the suspected disease, the complement will unite
with the antigen-amboceptor system and be “fixed,” that is, be no
longer capable of uniting with any other amboceptor-antigen system. No
chemical or physical means of telling whether this union has occurred
or not, except as given below, has been discovered as yet, though
doubtless will be by physico-chemical tests, nor can the combination
be seen. Hence an “indicator,” as is so frequently used in chemistry,
is put into the mixture of antigen-amboceptor-complement after it has
been allowed to stand in the incubator for one-half to one hour to
permit the union to become complete. The “indicator” used is a mixture
of sheep’s corpuscles and the heated (“inactivated”) blood serum of
a rabbit which has been injected with sheep’s blood corpuscles and
therefore contains a _hemolytic amboceptor specific_ for the corpuscles
which is capable also of uniting with complement. The indicator is
put into the first mixture and the whole is again incubated and
examined. If the mixture is _clear_ and _colorless_ with a _deposit
of red corpuscles_ at the bottom, that would mean that the complement
had been bound to the first complex, since it was not free to unite
with the second sheep’s corpuscles (antigen)--rabbit serum (hemolytic
amboceptor) complex--and destroy the corpuscles. Hence if the
complement is bound in the first instance, the _specific amboceptor_
for the first antigen must have been present in the blood, that is, the
animal was infected with the organism in question. Such a reaction is
called a “positive” test.
On the other hand, if the final solution is _clear_ but of a _red_
color, that would mean that complement must have united with the
corpuscles--hemolytic amboceptor system--and destroyed the corpuscles
in order to cause the _clear red_ solution of hemoglobin. If complement
united with this system it could not have united with the first system,
hence there was no _specific amboceptor_ there to bind it; no specific
amboceptor in the animal’s blood, means no infection. Hence a _red
solution_ is a “negative test.”
The scheme for the test may be outlined as follows:
Antigen + Patient’s Serum, heated + Complement
(specific for (unknown amboceptor) (derived from
the amboceptor guinea pig’s serum)
sought)
Incubate one-half hour in a water bath or one hour in an incubator.
Then add the indicator which is
Antigen + Amboceptor
(red blood corpuscles) (for corpuscles, serum of
a rabbit immunized against
the red corpuscles)
Incubate as above.
In practice all the different ingredients must be accurately tested,
standardized and used in exact quantities, and tests must also be run
as controls with a known normal blood of an animal of the same species
as the one examined and with a known positive blood.
It should be stated that in one variety of the Complement-Fixation
Test, namely, the “Wassermann Test for Syphilis” in human beings,
an antigen is used which is not derived from the specific organism
(_Treponema pallidum_) which causes the disease nor even from
syphilitic tissue. It has been determined that alcohol will extract
from certain tissues, _human or animal_, substances which _act
specifically_ in combining with the syphilitic amboceptor present
in the blood. Alcoholic extracts of beef heart are most commonly
used. Details of this test may be learned in the advanced course in
Immunity and Serum Therapy.
The complement-fixation test might be applied to the determination
of unknown bacteria, using the unknown culture as antigen and trying
it with the sera of different animals immunized against a variety
of organisms, some one of which might prove to furnish _specific
amboceptor_ for the unknown organism and hence indicate what it is. The
test used in this way has not been shown to be a practical necessity
and hence is rarely employed. It has been used, however, to detect
traces of unknown proteins, particularly blood-serum proteins, in
medico-legal cases in exactly the above outlined manner and is very
delicate and accurate.
CHAPTER XXX.
PHAGOCYTOSIS--OPSONINS.
It has been mentioned that Metchnikoff, in a publication in 1883,
attempted to explain immunity on a purely cellular basis. It has
been known since Haeckel’s first observation in 1858 that certain of
the white corpuscles do engulf solid particles that may get into the
body, and among them bacteria. Metchnikoff at first thought that this
engulfing and subsequent intracellular digestion of the microörganisms
were sufficient to protect the body from infection. The later
discoveries (discussed in considering Ehrlich’s theory of immunity)
of substances present in the blood serum and even in the blood plasma
which either destroy the bacteria or neutralize their action have
caused Metchnikoff to modify his theory to a great extent. He admitted
the presence of these substances, though giving them other names,
but ascribed their formation to the phagocytes or to the same organs
which form the leukocytes--lymphoid tissue generally, bone marrow. It
is not within the province of this work to attempt to reconcile these
theories, but it may be well to point out that Ehrlich’s theory is one
of _chemical substances_ and that the _origin_ of these substances is
not an _essential_ part of the theory, so that the two theories, except
in some minor details, are not necessarily mutually exclusive.
[Illustration: PLATE V
ELIE METCHNIKOFF]
Sir A. E. Wright and Douglas, in 1903, showed that even in those
instances where immunity depends on phagocytosis, as it certainly does
in many cases, the phagocytes are more or less inactive unless they are
aided by chemical substances present in the blood. These substances
_act on the bacteria, not on the leukocytes_, and change them in such
a way that they are more readily taken up by the phagocytes. Wright
proposed for these bodies the name _opsonin_, derived from a Greek
word signifying “to prepare a meal for.” Neufeld and Rimpau at about
the same time (1904), in studying immune sera, observed substances of
similar action in these sera and proposed the name _bacteriotropins_,
or bacteriotropic substances. There is scarcely a doubt that the two
names are applied to identical substances and that Wright’s name
_opsonin_ should have preference.
The chemical nature of opsonins is not certainly determined, but they
appear to be a distinct class of antibodies and to possess two groups,
a combining or haptophore and a preparing or opsonic group and hence
are similar to antibodies of Ehrlich’s second order--agglutinins and
precipitins. Wright also showed that opsonins are just as specific as
agglutinins are--that is, a micrococcus opsonin prepares micrococci
only for phagocytosis and not streptococci or any other bacteria.
Wright showed that opsonins for many bacteria are present in normal
serum and that in the serum of an animal which has been immunized
against such bacteria the opsonins are _increased_ in amount. Also
that in a person infected with certain bacteria the opsonins are
either increased or diminished, depending on whether the progress of
the infection is favorable or unfavorable. The _opsonic power_ of a
serum normal or otherwise is determined by mixing an emulsion of fresh
leukocytes in normal saline solution with a suspension of the bacteria
and with the serum to be tested. The leukocytes must first be washed in
several changes of normal salt solution to free them from any adherent
plasma or serum. The mixture is incubated for about fifteen minutes
and then slides are made, stained with a good differential blood
stain, Wright’s or other, and the average number of bacteria taken up
by at least fifty phagocytes taken in order in a field is determined
by counting under the microscope. The number so obtained Wright calls
the _phagocytic index_ of the serum tested. The phagocytic index of a
given serum divided by the phagocytic index of a normal serum gives
the _opsonic index_ of the serum tested. Assuming the normal opsonic
index to be 1, Wright asserts that in healthy individuals the range
should be not more than from 0.8 to 1.2, and that an index below 0.8
may show a great susceptibility for the organism tested, infection with
the given organism if derived from the individual, or improper dosage
in case attempts have been made to immunize by using killed cultures,
vaccines, of the organism.
On the occasion of the author’s visit to Wright’s clinic (1911) he
stated that he used the determination of the _opsonic index_ chiefly as
a _guide to the dosage_ in the use of vaccines.
Most workers outside the Wright school have failed to recognize any
essential value of determinations of the opsonic index in the use
of vaccines. Some of the reasons for this are as follows: The limit
of error in phagocytic counts may be as great as 50 per cent. in
different series of fifty, hence several hundred must be counted, which
adds greatly to the tediousness and time involved; the variation in
apparently healthy individuals is frequently great, hence the “normal”
is too uncertain; finally the opsonic index and the clinical course of
the disease do not by any means run parallel. Undoubtedly the method
has decided value in the hands of an individual who makes opsonic
determinations his chief work, as Wright’s assistants do, but it can
scarcely be maintained at the present time that such determinations
are necessary in vaccine therapy. Nevertheless that opsonins actually
exist and that they play an essential part in phagocytosis, and hence
in immunity, is now generally recognized.
BACTERIAL VACCINES.
Whether determinations of opsonic index are useful or not is largely
a matter of individual opinion, but there is scarcely room to doubt
that Wright has conferred a lasting benefit by his revival of the
use of _dead cultures of bacteria_, _bacterial vaccines_, both for
protective inoculation and for treatment. It is perhaps better to use
the older terms “vaccination” and “vaccine” (though the cow, _vacca_,
is not concerned) than to use Wright’s term “opsonic method” in this
connection, bearing in mind that the idea of a vaccine is that it
contains the _causative organism_ of the infection as indicated on p.
253.
As early as 1880 Touissant proposed the use of dead cultures of
bacteria to produce immunity. But because injections of such cultures
were so frequently followed by abscess formation, doubtless due to
the _high temperatures_ used to kill the bacteria, the method was
abandoned. Further, Pasteur and the French school persistently denied
the possibility of success with such a procedure, and some of them
even maintain this attitude at the present time. The successes of
Wright and the English school which are being repeated so generally
wherever properly attempted, leave no doubt in the unprejudiced of the
very great value of the method and have unquestionably opened a most
promising field both for preventive inoculation and for treatment in
many infectious diseases. That the practice is no more universally
applicable than are immune serums and that it has been and is still
being grossly overexploited is undoubted.
The use of a vaccine is based on two fundamental principles. The first
of these is that the cell introduced must not be in a condition to
cause serious injury to the animal by its multiplication and consequent
elaboration of injurious substances. The second is that, on the other
hand, it must contain antigens in such condition that they will act as
stimuli to the body cells to produce the necessary antibodies, whether
these be opsonins, bactericidal substances, or anti-endotoxins. In the
introduction of living organisms there is always more or less risk
of the organism not being sufficiently attenuated and hence of the
possibility of its producing too severe an infection. In using killed
cultures, great care must be exercised in destroying the organisms,
_so that the antigens are not at the same time rendered inactive_.
Hence in the preparation of bacterial vaccines by Wright’s method the
_temperature and the length of time used to kill the bacteria are most
important factors_. This method is in general to grow the organisms
on an agar medium, rub off the culture and emulsify in sterile normal
salt solution (0.85 per cent. NaCl). The number of bacteria per cc.
is determined by staining a slide made from a small volume of the
emulsion mixed with an equal volume of human blood drawn from the
finger and counting the relative number of bacteria and of red blood
corpuscles. Since the corpuscles are normally 5,000,000 per c.mm.,
a simple calculation gives the number of bacteria. The emulsion of
bacteria is then diluted so that a certain number of millions shall
be contained in each cc., “standardized” as it is called, then heated
to the proper temperature for the necessary time and it is ready for
use. A preservative, as 0.5 per cent. phenol, tricresol, etc., is added
unless the vaccine is to be used up at once. The amounts of culture,
salt solution, etc., vary with the purpose for which the vaccine is to
be used, from one or two agar slant cultures and a few cc. of solution,
when a single animal is to be treated, to bulk agar cultures and liters
of solution as in preparing antityphoid vaccine on a large scale.
Agar surface cultures are used so that there will be as little
admixture of foreign protein as possible (see Anaphylaxis, p. 289 _et
seq._). Normal saline solution is isotonic with the body cells and
hence is employed as the vehicle.
=Lipovaccines.=--The suspension of bacteria in neutral oil was first
used by Le Moignac and Pinoy who gave the name “lipovaccines” (λιπος
= fat) to them. It was claimed that the reaction following injection
of these vaccines was less severe than with saline vaccines in many
instances; also, that the bacteria were much more slowly absorbed. For
these two reasons it was hoped that much larger numbers of bacteria
could be injected at one dose and one injection would suffice instead
of three or more as ordinarily used. The technique of preparation,
standardization and killing of the organisms has not as yet been
sufficiently well established to warrant the general substitution of
lipovaccines for ordinary saline suspensions.
Vaccines are either “_autogenous_” or “_stock_.” An “autogenous”
vaccine is a vaccine that is made from bacteria derived from the
individual or animal which it is desired to vaccinate and contains
not only the particular organism but the particular strain of that
organism which is responsible for the lesion. Stock vaccines are
made up from organisms like the infective agent in a given case but
derived from some other person or animal or from laboratory cultures.
Commercial vaccines are “stock” vaccines and are usually “polyvalent”
or even “mixed.” A “polyvalent” vaccine contains several strains of the
infective agent and a “mixed” contains several different organisms.
Stock vaccines have shown their value when used as preventive
inoculations, notably so in typhoid fever in man, anthrax and black-leg
in cattle. The author is strongly of the opinion, not only from the
extended literature on the subject, but also from his own experience
in animal, and especially in human cases, that stock vaccines are
much inferior and much more uncertain in their action when used in
the _treatment_ of an infection, than are autogenous vaccines. This
applies particularly to those instances in which _pneumococci_,
_streptococci_, _micrococci_, and _colon bacilli_ are the causative
agents but to others as well. The following are some of the reasons for
this opinion: The above organisms are notoriously extremely variable in
their virulence. While there is no necessarily close connection between
virulence and antigenic property, yet since virulence is so variable,
it is rational to assume that antigenic property is also extremely
variable. Individuals vary just as much in susceptibility and hence in
reactive power, and generally speaking, an individual will react better
in the production of antibodies to a stimulus to which he has been more
or less subjected, _i.e._, to organisms derived from his own body.
In the preparation of a vaccine great care must be used in heating so
that the organisms are killed, but the _antigens_ are not destroyed.
Many of the enzymes present in bacteria, especially the proteolytic
ones, are not any more sensitive to heat than are the antigens, hence
are not destroyed entirely. Therefore a vaccine kept in stock for a
long time gradually has some of its antigens destroyed by the uninjured
enzymes present with them, and so loses in potency. Therefore in
treating a given infection it is well to make up a vaccine from the
lesion, use three or four doses and if more are necessary make up a new
vaccine.
If the above statements are borne in mind and vaccines are made and
administered accordingly, the author is well satisfied that much better
results will be secured.
In accordance with the theory on which the use of vaccines is based,
_i.e._, that they stimulate the body cells to produce immunizing
antibodies, it is clear that they are especially suitable in those
infections in which the process is _localized_ and should not be of
much value in _general_ infections. In the latter case the cells of
the body are stimulated to produce antibodies by the circulating
organisms, probably nearly to their limit, hence the introduction of
more of the same organisms, capable of stimulating though dead, is apt
to overtax the cells and do more harm than good. It is not possible to
tell accurately when this limit is reached, but the clinical symptoms
are a guide. If vaccines are used at all in general infections they
should be given in the early stages and in small doses at first with
close watch as to the effect. In localized infections only the cells in
the immediate neighborhood are much stimulated, hence the introduction
of a vaccine calls to their aid cells in the body generally, and much
more of the resulting antibodies are carried to the lesion in question.
Manifestly surgical procedures such as incision, drainage, washing
away of dead and necrotic tissue with normal saline solution, not
necessarily antiseptics, will aid the antibodies in their action and
are to be recommended where indicated.
In the practical application of any remedy the _dosage_ is most
important. Unfortunately there is no accurate method of determining
this with a vaccine. Wright recommended determining the number of
the organisms per cc. as before mentioned, and his method or some
modification of it is still in general use. From what was said with
regard to variation, both in organisms and in individuals, it can
be seen that the number of organisms is at least only a very rough
guide. This is further illustrated by the doses of micrococcus
(staphylococcus) vaccines recommended by different writers, which
vary from 50,000,000 to 2,000,000,000 per cc. The author is decidedly
of the opinion that _there is no way of determining the dosage of
a vaccine in the treatment of any given case except by the result
of the first dose_. Hence it is his practice to make vaccines of a
particular organism of the same approximate strength, and to give a
dose of a measured portion of a cubic centimeter, judging the amount
by what the individual or animal can apparently withstand, without too
violent a reaction. If there is no local or general reaction or if it
is very slight and there is no effect on the lesion, the dose is too
small. If there is a violent local reaction with severe constitutional
symptoms clinically, and the lesion appears worse, the dose is too
large. There should be some local reaction and some general, but not
enough to cause more than a slight disturbance, easy to judge in human
subjects, more difficult in animals. In cases suitable for vaccine
treatment no _serious_ results should follow from a properly prepared
vaccine, though the process of healing may be delayed temporarily.
Wright claimed, and many have substantiated him, that always following
a vaccination there is a period when the resistance of the animal is
diminished. This is called the “negative phase,” and Wright considered
this to last as long as the opsonic index remained low, and when this
latter began to increase the stage of the “positive” or favorable phase
was reached. As has been stated the opsonic index is pretty generally
regarded as of doubtful value, though the existence of a period of
lowered resistance is theoretically probable from the fact that
antibodies already present in the blood will be partially used up in
uniting with the vaccine introduced and that the body cells are called
upon suddenly to do an extra amount of work and it takes them some time
to adapt themselves. This time, the “negative phase,” is much better
determined by the clinical symptoms, general and especially local. It
is good practice to begin with a dose relatively small. The result
of this is an indication of the proper dosage and also prepares the
patient for a larger one. The second dose should follow the first not
sooner than three or four days, and should be five to seven days if the
first reaction is severe. These directions are not very definite, but
clinical experience to date justifies them. It is worth the time and
money to one who wishes to use vaccines to learn from one who has had
experience both in making and administering them, and then to remember
that each patient is an individual case, for the use of vaccines as
well as for any other kind of treatment.
AGGRESSIN.
Opsonins have been shown to be specific substances which act on
bacteria in such a way as to render them more readily taken up by the
leukocytes. By analogy one might expect to find bacteria secreting
specific substances which would tend to counteract the destructive
action of the phagocytes and bactericidal substances. Bail and his
co-workers claim to have demonstrated such substances in exudates in
certain diseases and have given the distinctive name “aggressins” to
them. By injecting an animal with “aggressins,” antiaggressins are
produced which counteract their effects and thus enable the bacteria to
be destroyed. The existence of such specific bodies is not generally
accepted as proved. The prevailing idea is that bacteria protect
themselves in any given case by the various toxic substances that they
produce, and that “aggressins” as a special class of substances are not
formed.
CHAPTER XXXI.
ANAPHYLAXIS.
Dallera, in 1874, and a number of physiologists of that period,
observed peculiar skin eruptions following the transfusion of blood,
that is, the introduction of foreign proteins. In the years subsequent
to the introduction of diphtheria antitoxin (1890) characteristic
“serum rashes” were not infrequently reported, sometimes accompanied by
more or less severe general symptoms and occasionally death--a train of
phenomena to which the name “serum sickness” was later applied, since
it was shown that it was the horse serum (foreign protein) that was
the cause, and not the antitoxin itself. In 1898 Richet and Hericourt
noticed that some of the dogs which they were attempting to immunize
against toxic eel serum not only were not immunized but suffered even
more severely after the second injection. They obtained similar results
with an extract of mussels which contain a toxin. Richet gave the name
“anaphylaxis” (“no protection”) to this phenomenon to distinguish it
from immunity or prophylaxis (protection).
All the above-mentioned observations led to no special investigations
as to their cause. In 1903, Arthus noticed abscess formation,
necrosis and sloughing following several injections of horse serum
in immediately adjacent parts of the skin in rabbits (“Arthus’
phenomenon”). Theobald Smith, in 1904, observed the death of
guinea-pigs following properly spaced injections of horse serum. This
subject was investigated by Otto and by Rosenau and Anderson in this
country and about the same time von Pirquet and Schick were making a
study of serum rashes mentioned above. The publications of these men
led to a widespread study of the subject of injections of foreign
proteins. It is now a well-established fact that the injection into an
animal of a foreign protein--vegetable, animal or bacterial, simple or
complex--followed by a second injection after a proper length of time
leads to a series of symptoms indicating poisoning, which may be so
severe as to cause the death of the animal. Richet’s term “anaphylaxis”
has been applied to the condition of the animal following the first
injection and indicates that it is in a condition of supersensitiveness
for the protein in question. The animal is said to be “sensitized”
for that protein.[25] The sensitization is specific since an animal
injected with white of chicken’s egg reacts to a second injection of
chicken’s egg only and not pigeon’s egg or blood serum or any other
protein. The specific poisonous substance causing the symptoms has
been called “anaphylotoxin” though what it is, is still a matter of
investigation. It is evident that some sort of an antibody results from
the first protein injected and that it is specific for its own antigen.
A period of ten days is usually the minimum time that must elapse
between the first and second injections in guinea-pigs in order that a
reaction may result, though a large primary dose requires much longer.
If the second injection is made within less time no effect follows,
and after three or more injections at intervals of about one week the
animal fails to react at all, it has become “immune” to the protein.
Furthermore, after an animal has been sensitized by one injection and
has reacted to a second, then, if it does not die from the reaction, it
fails to react to subsequent injections. In this latter case it is said
to be “antianaphylactic.”
It must be remembered that proteins do not normally get into the
circulation except by way of the alimentary tract. Here all proteins
that are absorbed are first broken down to their constituent
amino-acids, absorbed as such and these are built up into the proteins
characteristic of the animal’s blood. Hence when protein as such gets
into the blood it is a foreign substance to be disposed of. The blood
contains proteolytic enzymes for certain proteins normally. It is
also true that the body cells possess the property of digesting the
proteins of the blood and building them up again into those which are
characteristic of the cell. Hence it appears rational to assume that
the foreign proteins act as stimuli to certain cells to produce more
of the enzymes necessary to decompose them, so that they may be either
built up into cell structure or eliminated as waste. If in this process
of splitting up of protein a poison were produced, then the phenomena
of “anaphylaxis” could be better understood. As a matter of fact
Vaughan and his co-workers have shown that by artificially splitting
up proteins from many different sources--animal, vegetable, pathogenic
and saprophytic bacteria--a poison _is produced_ which appears to be
the same in all cases and which causes the symptoms characteristic of
anaphylaxis. On the basis of these facts it is seen that anaphylaxis
is simply another variety of immunity. The _specific antibody_ in
this case is an _enzyme_ which decomposes the protein instead of
precipitating it. The enzyme must be specific for the protein since
these differ in constitution. Vaughan even goes so far as to say that
the poison is really the central ring common to all proteins and that
they differ only in the lateral groups or side chains attached to this
central nucleus. The action of the enzyme in this connection would be
to split off the side chains, and since these are the specific parts of
the protein, the enzyme must be specific for each protein. The pepsin
of the gastric juice and the trypsin of the pancreas split the native
proteins only to peptones. As is well known, these when injected in
sufficient quantity give rise to poisonous symptoms, and will also
give rise to anaphylaxis under properly spaced injections. They do not
poison normally because they are split by the intestinal erepsin to
amino-acids and absorbed as such. Whether Vaughan’s theory of protein
structure is the true one or not remains to be demonstrated. It is
not essential to the theory of anaphylaxis above outlined, _i.e._,
a phenomenon due to the action of specific _antibodies_ which are
enzymes. On physiological grounds this appears the most rational of the
few explanations of anaphylaxis that have been offered and was taught
by the author before he had read Vaughan’s theory along the same lines.
On the basis of the author’s theory the phenomena of protein immunity
and antianaphylaxis may be explained in the following way which the
author has not seen presented. The enzymes necessary to decompose the
injected protein are present in certain cells and are formed in larger
amount by those cells to meet the increased demand due to injection
of an excess of protein. They are retained in the cell for a time at
least. If a second dose of protein is given before the enzymes are
excreted from the cells as waste, this is digested within the cells in
the normal manner. If a third dose is given, the cells adapt themselves
to this increased intracellular digestion and it thus becomes normal
to them. Hence the _immunity_ is due to this increased intracellular
digestion.
On the other hand, if the second injection is delayed long enough, then
the _excess_ enzyme, but not all, is excreted from the cells and meets
the second dose of protein in the blood stream and rapidly decomposes
it there, so that more or less intoxication from the split products
results. This uses up _excess_ enzyme, hence subsequent injections are
not digested in the blood stream but within the cells as before. So
that “antianaphylaxis” is dependent on the exhaustion of the excess
enzyme in the blood, and the condition is _fundamentally_ the same as
protein immunity, _i.e._, due to _intracellular_ digestion in each case.
As has been indicated “serum sickness” and sudden death following
serum injections are probably due to a sensitization of the individual
to the proteins of the horse in some unknown way. Probably hay fever
urticarial rashes and idiosyncrasies following the ingestion of certain
foods--strawberries, eggs, oysters, etc., are anaphylactic phenomena.
In medical practice the reaction is used as a means of diagnosis in
certain diseases, such as the tuberculin test in tuberculosis, the
mallein test in glanders. The individual or animal with tuberculosis
becomes sensitized to certain proteins of the tubercle bacillus and
when these proteins in the form of tuberculin are introduced into the
body a reaction results, local or general, according to the method of
introduction. The practical facts in connection with the tuberculin
test are also in harmony with the author’s theory of anaphylaxis
as above outlined. Milder cases of tuberculosis give more vigorous
reactions because the intracellular enzymes are not used up rapidly
enough since the products of the bacillus are secreted slowly in such
cases. Hence excess of enzyme is free in the blood and the injection of
the tuberculin meets it there and a vigorous reaction results. In old,
far-advanced cases, no reaction occurs, because the enzymes are all
used in decomposing the large amount of tuberculous protein constantly
present in the blood. The fact that an animal which has once reacted
fails to do so until several months afterward likewise depends on the
fact that the _excess_ enzyme is used in the reaction and time must
elapse for a further excess to accumulate.
The anaphylactic reaction has been made use of in the identification of
various types of proteins and is of very great value since the reaction
is so delicate, particularly when guinea-pigs are used as test animals.
Wells has detected the 0.000,001 g. of protein by this test. It is
evident that the test is applicable in medico-legal cases and in food
examination and has been so used.
A TABULATION OF ANTIGENS AND ANTIBODIES AS AT PRESENT RECOGNIZED.
CLASS OF
ANTIGEN ANTIBODY ACTION OF ANTIBODY RECEPTOR
Toxin Antitoxin Combines with toxin and I.
hence prevents toxin
from uniting with a
cell and injuring it,
_i.e._, neutralizes toxin.
Enzyme Antienzyme Combines with enzyme I.
and thus prevents enzyme
from uniting
with anything else and
showing its action, _i.e._,
neutralizes enzyme.
Solution of Precipitin Unites with its antigen II.
protein and causes its precipitation
from solution.
Solution of ? Causes phenomenon of (?)
protein anaphylaxis(?)
Suspension of Agglutinin Unites with its antigen II.
cells causes its clumping together
and settling out
of suspension.
Suspension of Opsonin Unites with its antigen II.
cells and makes the cells (?)
more easily taken up
by phagocytes.
Suspension of Amboceptor Unites with its antigen III.
cells and also with complement
which latter then
dissolves the antigen.
Precipitin Antiprecipitin Neutralizes precipitin. I.
Agglutinin Antiagglutinin Neutralizes agglutinin. I.
Opsonin Antiopsonin Neutralizes opsonin. I.
Amboceptor Antiamboceptor Neutralizes amboceptor. I.
(two kinds)
Complement Anticomplement Neutralizes complement. I.
SUMMARY OF IMMUNITY AS APPLIED TO PROTECTION FROM DISEASE.
The discussion of “immunity problems” in the preceding chapters serves
to show that protection from disease either as a condition natural to
the animal or as an acquired state is dependent on certain properties
of its body cells or fluids, or both. The actual factors so far as at
present known may be summarized as follows:
1. _Antitoxins_ which neutralize true toxins; shown to exist for very
few diseases.
2. _Cytolytic substances_ which destroy the invading organism: in
reality two substances; amboceptor, which is specific, and complement,
the real dissolving enzyme.
3. _Phagocytosis_ or the destruction of the invading organisms within
the leukocytes.
4. _Opsonins_ which render the bacteria more readily taken up by the
phagocytes.
5. _Enzymes_ other than complement possibly play a part in the
destruction of some pathogenic organisms or their products. This
remains to be more definitely established.
6. It is possible that in natural immunity there might be no receptors
in the body cells to take up the organisms or their products, or the
receptors might be present in certain cells but of a very low chemical
affinity, so that combination does not occur. It is even highly
probable that many substances formed by invading organisms which might
injure specialized cells, such as those of glandular, nervous or muscle
tissue, have a more rapid rate of reaction with, or a stronger affinity
for, lower unspecialized cells, such as connective and lymphoid tissue,
and unite with these so that their effects are not noticed.
The importance of these different, factors varies in different diseases
and need not be considered in this connection.
The question “which of the body cells are engaged in the production
of antibodies” is not uncommonly asked. On physiological grounds it
would not seem reasonable that the highly specialized tissues above
mentioned could take up this work, even though they are the ones which
suffer the greatest injury in disease. Hence it is to be expected that
the lower or unspecialized cells are the source, and it has been shown
that the antibodies are produced by the phagocytes (though not entirely
as Metchnikoff maintained), by lymphoid tissue generally, by the bone
marrow and also by connective-tissue cells, though in varying degrees.
Since immunity depends on the activity of the body cells it is evident
that one of the very best methods for avoiding infectious diseases is
to keep these cells up to their highest state of efficiency, to keep in
“good health.” Hence good health means not only _freedom from disease_
but also _protection against disease_.
LIST OF LABORATORY EXERCISES GIVEN IN CONNECTION WITH THE CLASS WORK
INCLUDED IN THIS TEXT-BOOK.
Exercise 1. Cleaning glassware.
Exercise 2. Preparation of broth medium from meat juice.
Exercise 3. Preparation of gelatin medium from broth.
Exercise 4. Preparation of agar medium from broth.
Exercise 5. Potato tubes.
Exercise 6. Potato plates.
Exercise 7. Plain milk tubes.
Exercise 8. Litmus milk tubes.
Exercise 9. Sugar broth media.
Exercise 10. Blood-serum tubes.
Exercise 11. Inoculation of tubes. Action on complex proteins.
Exercise 12. Production of gas from carbohydrates.
Exercise 13. Production of indol.
Exercise 14. Reduction of nitrates.
Exercise 15. Chromogenesis: Illustrates nicely the variation with
environment.
Exercise 16. Enzyme production.
Exercise 17. Making of plate cultures; isolation in pure culture.
Exercise 18. Stain making and staining.
Exercise 19. Cell forms and cell groupings.
Exercise 20. Hanging drop slides.
Exercise 21. Staining of spores.
Exercise 22. Staining of acid-fast bacteria.
Exercise 23. Staining of capsules.
Exercise 24. Staining of metachromatic granules.
Exercise 25. Staining of flagella.
Exercise 26. Study of individual species.
Exercise 27. Determination of thermal death-point.
Exercise 28. Action of disinfectants on bacteria.
Exercise 29. Action of sunlight on bacteria.
DESCRIPTIVE CHART--SOCIETY OF AMERICAN BACTERIOLOGISTS.
_Prepared by Committee on Methods of Identification of Bacterial
Species.--F. D. Chester, F. P. Gorham, Erwin F. Smith._
_Endorsed by the Society for general use at the Annual Meeting,
December, 1907._
GLOSSARY OF TERMS.
AGAR HANGING BLOCK, a small block of nutrient agar cut from a pour
plate, and placed on a cover-glass, the surface next the glass having
been first touched with a loop from a young fluid culture or with a
dilution from the same. It is examined upside down, the same as a
hanging drop.
AMEBOID, assuming various shapes like an ameba.
AMORPHOUS, without visible differentiation in structure.
ARBORESCENT, a branched, tree-like growth.
BEADED, in stab or stroke, disjointed or semiconfluent colonies along
the lines of inoculation.
BRIEF, a few days, a week.
BRITTLE, growth dry, friable under the platinum needle.
BULLATE, growth rising in convex prominences, like a blistered surface.
BUTYROUS, growth of a butter-like consistency.
CHAINS,
Short chains, composed of 2 to 8 elements.
Long chains, composed of more than 8 elements.
CILIATE, having fine, hair-like extensions, like cilia.
CLOUDY, said of fluid cultures which do not contain pseudozoogleæ.
COAGULATION,[22] the separation of casein from whey in milk. This may
take place quickly or slowly, and as the result either of the formation
of an acid or of a lab ferment.
CONTOURED, an irregular, smoothly undulating surface, like that of a
relief map.
CONVEX surface, the segment of a circle, but flattened.
COPROPHYL, dung bacteria.
CORIACEOUS, growth tough, leathery, not yielding to the platinum needle.
CRATERIFORM, round, depressed, due to the liquefaction of the medium.
CRETACEOUS, growth opaque and white, chalky.
CURLED, composed of parallel chains in wavy strands, as in anthrax
colonies.
DIASTASIC ACTION, same as DIASTATIC, conversion of starch into
water-soluble substances by diastase.
ECHINULATE, in agar stroke a growth along line of inoculation, with
toothed or pointed margins; in stab cultures growth beset with pointed
outgrowths.
EFFUSE, growth thin, veily, unusually spreading.
ENTIRE, smooth, having a margin destitute of teeth or notches.
EROSE, border irregularly toothed.
FILAMENTOUS, growth composed of long, irregularly placed or interwoven
filaments.
FILIFORM, in stroke or stab cultures a uniform growth along line of
inoculation.
FIMBRIATE, border fringed with slender processes, larger than filaments.
FLOCCOSE, growth composed of short curved chains, variously oriented.
FLOCCULENT, said of fluids which contain pseudozoogleæ, _i.e._, small
adherent masses of bacteria of various shapes and floating in the
culture fluid.
FLUORESCENT, having one color by transmitted light and another by
reflected light.
GRAM’S STAIN, a method of differential bleaching after gentian violet,
methyl violet, etc. The + mark is to be given only when the bacteria
are deep blue or remain blue after counter-staining with Bismarck brown.
GRUMOSE, clotted.
INFUNDIBULIFORM, form of a funnel or inverted cone.
IRIDESCENT, like mother-of-pearl. The effect of very thin films.
LACERATE, having the margin cut into irregular segments as if torn.
LOBATE, border deeply undulate, producing lobes (see _Undulate_).
LONG, many weeks, or months.
MAXIMUM TEMPERATURE, temperature above which growth does not take place.
MEDIUM, nutrient substance upon which bacteria are grown.
MEMBRANOUS, growth thin, coherent, like a membrane.
MINIMUM TEMPERATURE, temperature below which growth does not take place.
MYCELIOID, colonies having the radiately filamentous appearance of mold
colonies.
NAPIFORM, liquefaction with the form of a turnip.
NITROGEN REQUIREMENTS, the necessary nitrogenous food. This is
determined by adding to _nitrogen-free_ media the nitrogen compound to
be tested.
OPALESCENT, resembling the color of an opal.
OPTIMUM TEMPERATURE, temperature at which growth is most rapid.
PELLICLE, in fluid bacterial growth forming either a continuous or an
interrupted sheet over the fluid.
PEPTONIZED, said of curds dissolved by trypsin.
PERSISTENT, many weeks, or months.
PLUMOSE, a fleecy or feathery growth.
PSEUDOZOOGLEÆ, clumps of bacteria, not dissolving readily in water,
arising from imperfect separation, or more or less fusion of the
components, but not having the degree of compactness and gelatinization
seen in zoogleæ.
PULVINATE, in the form of a cushion, decidedly convex.
PUNCTIFORM, very minute colonies, at the limit of natural vision.
RAPID, developing in twenty-four to forty-eight hours.
RAISED, growth thick, with abrupt or terraced edges.
RHIZOID, growth of an irregular branched or root-like character, as in
_B. mycoides_.
RING, same as RIM, growth at the upper margin of a liquid culture,
adhering more or less closely to the glass.
REPAND, wrinkled.
SACCATE, liquefaction the shape of an elongated sac, tubular,
cylindrical.
SCUM, floating islands of bacteria, an interrupted pellicle or bacteria
membrane.
SLOW, requiring five or six days or more for development.
SHORT, applied to time, a few days, a week.
SPORANGIA, cells containing endospores.
SPREADING, growth extending much beyond the line of inoculation,
_i.e._, several millimetres or more.
STRATIFORM, liquefying to the walls of the tube at the top and then
proceeding downward horizontally.
THERMAL DEATH-POINT, the degree of heat required to kill young fluid
cultures of an organism exposed for ten minutes (in thin-walled
test-tubes of a diameter not exceeding 20 mm.) in the thermal
water-bath. The water must be kept agitated so that the temperature
shall be uniform during the exposure.
TRANSIENT, a few days.
TURBID, cloudy with flocculent particles; cloudy plus flocculence.
UMBONATE, having a button-like, raised centre.
UNDULATE, border wavy, with shallow sinuses.
VERRUCOSE, growth wart-like, with wart-like prominences.
VERMIFORM-CONTOURED, growth like a mass of worms or intestinal coils.
VILLOUS, growth beset with hair-like extensions.
VISCID, growth follows the needle when touched and withdrawn, sediment
on shaking rises as a coherent swirl.
ZOOGLEÆ, firm gelatinous masses of bacteria, one of the most typical
examples of which is the _Streptococcus mesenterioides_ of sugar vats.
(_Leuconostoc mesenterioides_), the bacterial chains being surrounded
by an enormously thickened, firm covering inside of which there may be
one or many groups of the bacteria.
NOTES.
(1) For decimal system of group numbers see Table I. This will be found
useful as a quick method of showing close relationships inside the
genus, but is not a sufficient characterization of any organism.
(2) The morphological characters shall be determined and described
from growths obtained upon at least one solid medium (nutrient agar)
and in at least one liquid medium (nutrient broth). Growths at 37° C.
shall be in general not older than twenty-four to forty-eight hours,
and growths at 20° C. not older than forty-eight to seventy-two hours.
To secure uniformity in cultures, in all cases preliminary cultivation
shall be practised as described in the revised Report of the Committee
on Standard Methods of the Laboratory Section of the American Public
Health Association, 1905.
(3) The observation of cultural and biochemical features shall cover
a period of at least fifteen days and frequently longer, and shall be
made according to the revised Standard Methods above referred to. All
media shall be made according to the same Standard Methods.
(4) Gelatin stab cultures shall be held for six weeks to determine
liquefaction.
(5) Ammonia and indol tests shall be made at end of tenth day, nitrite
tests at end of fifth day.
(6) Titrate with N/20 NaOH, using phenolphthalein as an indicator; make
titrations at same time from blank. The difference gives the amount of
acid produced.
The titration should be done after boiling to drive off any CO₂ present
in the culture.
(7) Generic nomenclature shall begin with the year 1872 (Cohn’s first
important paper).
Species nomenclature shall begin with the year 1880 (Koch’s discovery
of the pour plate method for the separation of organisms).
(8) Chromogenesis shall be recorded in standard color terms.
TABLE I.
A NUMERICAL SYSTEM OF RECORDING THE SALIENT CHARACTERS OF AN ORGANISM.
(GROUP NUMBER.)
100 Endospores produced
200 Endospores not produced
10 Aërobic (strict)
20 Facultative anaërobic
30 Anaërobic (strict)
1 Gelatin liquefied
2 Gelatin not liquefied
0.1 Acid and gas from dextrose
0.2 Acid without gas from dextrose
0.3 No acid from dextrose
0.4 No growth with dextrose
0.01 Acid and gas from lactose
0.02 Acid without gas from lactose
0.03 No acid from lactose
0.04 No growth with lactose
0.001 Acid and gas from saccharose
0.002 Acid without gas from saccharose
0.003 No acid from saccharose
0.004 No growth with saccharose
0.0001 Nitrates reduced with evolution of gas
0.0002 Nitrates not reduced
0.0003 Nitrates reduced without gas formation
0.00001 Fluorescent
0.00002 Violet chromogens
0.00003 Blue chromogens
0.00004 Green chromogens
0.00005 Yellow chromogens
0.00006 Orange chromogens
0.00007 Red chromogens
0.00008 Brown chromogens
0.00009 Pink chromogens
0.00000 Non-chromogenics
0.000001 Diastasic action on potato starch, strong
0.000002 Diastasic action on potato starch, feeble
0.000003 Diastasic action on potato starch, absent
0.0000001 Acid and gas from glycerin
0.0000002 Acid without gas from glycerin
0.0000003 No acid from glycerin
0.0000004 No growth with glycerin
The genus according to the system of Migula is given its proper symbol
which precedes the number thus:(7)
BACILLUS COLI (Esch.) Mig. becomes B. 222.111102
BACILLUS ALCALIGENES Petr. becomes B. 212.333102
PSEUDOMONAS CAMPESTRIS (Pam.) Sm. becomes Ps. 211.333151
BACTERIUM SUICIDA Mig. becomes Bact. 222.232103
Source............ Date of Isolation.............. Name........
Group No.(1)...............
DETAILED FEATURES.
NOTE--Underscore required terms. Observe notes and glossary of terms on
opposite side of card.
I. MORPHOLOGY(2)
1. Vegetative Cells, Medium used.............................
temp....................age.................days
Form, _round_, _short rods_, _long rods_, _short chains_, _long
chains_, _filaments_, _commas_, _short spirals_, _long spirals_,
_clostridium_, _cuneate_, _clavate_, _curved_.
Limits of Size..........................
Size of Majority.............................
Ends, _rounded_, _truncate_, _concave_.
{Orientation (grouping)............................
Agar {Chains (No. of elements)........................
Hanging-block {_Short chains_, _long chains_
{Orientation of chains, _parallel_, _irregular_.
2. Sporangia, medium
used.....................temp..............age..............days
Form, _elliptical_, _short rods_, _spindled_, _clavate_, _drumsticks_.
Limits of Size................
Size of Majority..............
Agar {Orientation (grouping)........
Hanging-block {Chains (No. of elements)......
{Orientation of chains, _parallel_, _irregular_.
Location of Endospores, _central_, _polar_.
3. Endospores.
Form, _round_, _elliptical_, _elongated_.
Limits of Size................
Size of Majority..............
Wall, _thick_, _thin_.
Sporangium wall, _adherent_, _not adherent_.
Germination, _equatorial_, _oblique_, _polar_, _bipolar_, _by
stretching_.
4. Flagella, No........Attachment _polar_, _bipolar_,
_peritrichiate_. How Stained.........
5. Capsules, present on.............
6. Zooglea, Pseudozooglea.
7. Involution Forms, on........in.....days at....° C.
8. Staining Reactions.
1:10 watery fuchsin, gentian violet, carbol-fuchsin, Loeffler’s
alkaline methylene blue.
Special Stains.
Gram....................Glycogen...............
Fat.....................Acid-fast................
Neisser.................
II. CULTURAL FEATURES(3)
1. Agar Stroke.
Growth, _invisible_, _scanty_, _moderate_, _abundant_.
Form of growth, _filiform_, _echinulate_, _beaded_, _spreading_,
_plumose_, _arborescent_, _rhizoid_.
Elevation of growth, _flat_, _effuse_, _raised_, _convex_.
Lustre, _glistening_, _dull_, _cretaceous_.
Topography, _smooth_, _contoured_, _rugose_, _verrucose_.
Optical characters, _opaque_, _translucent_, _opalescent_,
_iridescent_.
Chromogenesis(3)................
Odor, _absent_, _decided_, _resembling_............
Consistency, _slimy_, _butyrous_, _viscid_, _membranous_,
_coriaceous_, _brittle_.
Medium _grayed_, _browned_, _reddened_, _blued_, _greened_.
2. Potato.
Growth _scanty_, _moderate_, _abundant_, _transient_, _persistent_.
Form of growth, _filiform_, _echinulate_, _beaded_, _spreading_,
_plumose_, _arborescent_, _rhizoid_.
Elevation of growth, _flat_, _effuse_, _raised_, _convex_.
Lustre, _glistening_, _dull_, _cretaceous_.
Topography, _smooth_, _contoured_, _rugose_, _verrucose_.
Chromogenesis(3)...........Pigment in water _insoluble_, _soluble_:
other solvents.....................
Odor, _absent_, _decided_, _resembling_....................
Consistency, _slimy_, _butyrous_, _viscid_, _membranous_,
_coriaceous_, _brittle_.
Medium, _grayed_, _browned_, _reddened_, _blued_, _greened_.
3. Loeffler’s Blood-serum.
Stroke _invisible_, _scanty_, _moderate_, _abundant_.
Form of growth, _filiform_, _echinulate_, _beaded_, _spreading_,
_plumose_, _arborescent_, _rhizoid_.
Elevation of growth, _flat_, _effuse_, _raised_, _convex_.
Lustre, _glistening_, _dull_, _cretaceous_.
Topography, _smooth_, _contoured_, _rugose_, _verrucose_.
Chromogenesis(3)..........................
Medium _grayed_, _browned_, _reddened_, _blued_, _greened_.
Liquefaction begins in.............d, complete in................d,
4. Agar Stab.
Growth _uniform_, _best at top_, _best at bottom_: surface growth
_scanty_, _abundant_: _restricted_, _wide-spread_.
Line of puncture, _filiform_, _beaded_, _papillate_, _villous_,
_plumose_, _arborescent_: _liquefaction_.
5. Gelatin Stab.
Growth uniform, _best at top_, _best at bottom_.
Line of puncture, _filiform_, _beaded_, _papillate_, _villous_,
_plumose_, _arborescent_.
Liquefaction _crateriform_, _napiform_, _infundibuliform_,
_saccate_, _stratiform_: begins in....................d. complete
in....................d
Medium _fluorescent_, _browned_...............
6. Nutrient Broth.
Surface growth, _ring_, _pellicle_, _flocculent_, _membranous_,
_none_.
Clouding _slight_, _moderate_, _strong_: _transient_, _persistent_:
_none_: _fluid turbid_.
Odor, _absent_, _decided_, _resembling_..................
Sediment, _compact_, _flocculent_, _granular_, _flaky_, _viscid on
agitation_, _abundant_, _scant_.
7. Milk.
Clearing without coagulation.
Coagulation _prompt_, _delayed_, _absent_.
Extrusion of whey begins in............days.
Coagulum _slowly peptonized_, _rapidly peptonized_.
Peptonization begins on....d, complete on ....d.
Reaction, 1d...., 2d...., 4d...., 10d...., 20d....
Consistency, _slimy_, _viscid_, _unchanged_.
Medium _browned_, _reddened_, _blued_, _greened_.
Lab ferment, _present_, _absent_.
8. Litmus Milk.
_Acid_, _alkaline_, _acid then alkaline_, _no change_.
_Prompt reduction_, _no reduction_, _partial slow reduction_.
9. Gelatin Colonies.
Growth _slow_, _rapid_.
Form, _punctiform_, _round_, _irregular_, _ameboid_, _mycelioid_,
_filamentous_, _rhizoid_.
Elevation, _flat_, _effuse_, _raised_, _convex_, _pulvinate_,
_crateriform_ (_liquefying_).
Edge, _entire_, _undulate_, _lobate_, _erose_, _lacerate_,
_fimbriate_, _filamentous_, _floccose_, _curled_.
Liquefaction, _cup_, _saucer_, _spreading_.
10. Agar Colonies.
Growth _slow_, _rapid_ (temperature..............)
Form, _punctiform_, _round_, _irregular_, _ameboid_, _mycelioid_,
_filamentous_, _rhizoid_.
Surface _smooth_, _rough_, _concentrically ringed_, _radiate_,
_striate_.
Elevation, _flat_, _effuse_, _raised_, _convex_, _pulvinate_,
_umbonate_.
Edge, _entire_, _undulate_, _lobate_, _erose_, _lacerate_,
_fimbriate_, _floccose_, _curled_.
Internal structure, _amorphous_, _finely_, _coarsely granular_,
_grumose_, _filamentous_, _floccose_, _curled_.
11. Starch Jelly.
Growth, _scanty_, _copious_.
Diastatic action, _absent_, _feeble_, _profound_.
Medium stained...................
12. Silicate Jelly (Fermi’s Solution).
Growth _copious_, _scanty_, _absent_.
Medium stained..................
13. Cohn’s Solution.
Growth _copious_, _scanty_, _absent_.
Medium _fluorescent_, _non-fluorescent_.
14. Uschinsky’s Solution.
Growth _copious_, _scanty_, _absent_.
Fluid _viscid_, _not viscid_.
15. Sodium Chloride in Bouillon.
Per cent. inhibiting growth........................
16. Growth in Bouillon over Chloroform, _unrestrained_,
_feeble_, _absent_.
17. Nitrogen. Obtained from _peptone_, _asparagin_, _glycocoll_,
_urea_, _ammonia salts_, _nitrogen_.
18. Best media for long-continued growth...................
.....................................................
19. Quick tests for differential purposes..................
.....................................................
.....................................................
III. PHYSICAL AND BIOCHEMICAL FEATURES.
+----------------------------------+---+---+---+---+---+---+---+---+
| | D | S | L | M | G | M | | |
| | e | a | a | a | l | a | | |
| | x | c | c | l | y | n | | |
| | t | c | t | t | c | n | | |
| 1. Fermentation-tubes containing | r | h | o | o | e | i | | |
| peptone-water or | o | a | s | s | r | t | | |
| sugar-tree bouillon and | s | r | e | e | i | | | |
| | e | o | | | n | | | |
| | | s | | | | | | |
| | | e | | | | | | |
+----------------------------------+---+---+---+---+---+---+---+---+
| Gas production, in per cent. | | | | | | | | |
+----------------------------------+---+---+---+---+---+---+---+---+
| (H/CO₂) | | | | | | | | |
+----------------------------------+---+---+---+---+---+---+---+---+
| Growth in closed arm | | | | | | | | |
+----------------------------------+---+---+---+---+---+---+---+---+
| Amount of acid produced 1d. | | | | | | | | |
+----------------------------------+---+---+---+---+---+---+---+---+
| Amount of acid produced 2d. | | | | | | | | |
+----------------------------------+---+---+---+---+---+---+---+---+
| Amount of acid produced 3d. | | | | | | | | |
+----------------------------------+---+---+---+---+---+---+---+---+
2. Ammonia production, _feeble_, _moderate_, _strong_, _absent_,
_masked by acids_.
3. Nitrates in nitrate broth.
_Reduced_, _not reduced_.
Presence of nitrites...........ammonia..................
Presence of nitrates...........free nitrogen............
4. Indol production, _feeble_, _moderate_, _strong_.
5. Toleration of Acids, _great_, _medium_, _slight_.
_Acids tested_..............
6. Toleration of NaOH, _great_, _medium_, _slight_.
7. Optimum reaction for growth in bouillon, stated in terms of
Fuller’s scale..........................
8. Vitality on culture media, _brief_, _moderate_, _long_.
9. Temperature relations.
Thermal death-point (10 minutes’ exposure in nutrient broth when this
is adapted to growth of organism)............C.
Optimum temperature for growth......° C.; or best growth at 16° C.,
20° C., 25° C., 30° C., 37° C., 40° C., 50° C., 60° C.
Maximum temperature for growth.......... ° C.
Minimum temperature for growth.......... ° C.
10. Killed readily by drying: resistant to drying.
11. Per cent. killed by freezing (salt and crushed ice or liquid
air)................
12. Sunlight: Exposure on ice in thinly sown agar plates; one-half
plate covered (time 15 minutes), _sensitive_, _not sensitive_.
Per cent. killed................
13. Acids produced.................
14. Alkalies produced...............
15. Alcohols.......................
16. Ferments, _pepsin_, _trypsin_, _diastase_, _invertase_,
_pectase_, _cytase_, _tyrosinase_, _oxidase_, _peroxidase_,
_lipase_, _catalase_, _glucase_, _galactase_, _lab_,
_etc._........................
17. Crystals formed:.....
18. Effect of germicides:
+-----------+-------------+---+---+---+---+---+
| | | M | T | K | A | r |
| | | i | e | i | m | e |
| | | n | m | l | t | s |
| | | u | p | l | . | t |
| | | t | e | i | | r |
| | | e | r | n | r | a |
| | | s | a | g | e | i |
| Substance | Method used | | t | | q | n |
| | | | u | q | u | |
| | | | r | u | i | g |
| | | | e | a | r | r |
| | | | | n | e | o |
| | | | | t | d | w |
| | | | | i | | t |
| | | | | t | t | h |
| | | | | y | o | |
| | | | | | | |
+-----------+-------------+---+---+---+---+---+
| | | | | | | |
+-----------+-------------+---+---+---+---+---+
| | | | | | | |
+-----------+-------------+---+---+---+---+---+
| | | | | | | |
+-----------+-------------+---+---+---+---+---+
| | | | | | | |
+-----------+-------------+---+---+---+---+---+
| | | | | | | |
+-----------+-------------+---+---+---+---+---+
| | | | | | | |
+-----------+-------------+---+---+---+---+---+
| | | | | | | |
+-----------+-------------+---+---+---+---+---+
| | | | | | | |
+-----------+-------------+---+---+---+---+---+
| | | | | | | |
+-----------+-------------+---+---+---+---+---+
IV. PATHOGENICITY.
1. Pathogenic to Animals.
_Insects_, _crustaceans_, _fishes_, _reptiles_, _birds_, _mice_,
_rats_, _guinea-pigs_, _rabbits_, _dogs_, _cats_, _sheep_, _goats_,
_cattle_, _horses_, _monkeys_, _man_..........................
2. Pathogenic to Plants:
.........................................................
.........................................................
.........................................................
3. Toxins, _soluble_, _endotoxins_.
4. Non-toxin forming.
5. Immunity bactericidal.
6. Immunity non-bactericidal.
7. Loss of virulence on culture-media: _prompt_, _gradual_, _not
observed in_.....................months.
+-------------------------------------+
| BRIEF CHARACTERIZATION. |
| |
| Mark + or 0, and when two terms |
| occur on a line erase the one which |
| does not apply unless both apply. |
| |
+--------------------------------+----+
| M | Diameter over 1µ |----|
| O | Chains, filaments |----|
| R | Endospores |----|
| P | Capsules |----|
| H | Zooglea, Pseudozooglea |----|
| O | Motile |----|
| L | Involution forms |----|
| O | Gram’s stain |----|
| G | |----|
| Y | |----|
|(2)| |----|
+---+----------------------------+----+
| C | B | Cloudy, turbid |----|
| U | r | Ring |----|
| L | o | Pellicle |----|
| T | t | Sediment |----|
| U | h | |----|
| R +-----+----------------------+----+
| A | A | Shining |----|
| L | g | Dull |----|
| | a | Wrinkled |----|
| F | r | Chromogenic |----|
| E +-----+----------------------+----+
| A | G | Round |----|
| T | e | Proteus-like |----|
| U | l. | Rhizoid |----|
| R | | Filamentous |----|
| E | P | Curled |----|
| S | l | |----|
|(3)| a | |----|
| | t | |----|
| | e | |----|
| +-----+----------------------+----+
| | G S | Surface growth |----|
| | e t | Needle growth |----|
| | l a | |----|
| | . b.| |----|
| +-----+----------------------+----+
| | P | Moderate, absent |----|
| | o | Abundant |----|
| | t | Discolored |----|
| | a | Starch destroyed |----|
| | t | |----|
| | o | |----|
| +-----+---------------------------+
| | Grows at 37° C. |----|
| | Grows in Cohn’s sol. |----|
| | Grows in Uschinsky’s sol. |----|
|---+-----+----------------------+----+
| B | L f | Gelatin(4) |----|
| I | i a | Blood-serum |----|
| O | q c | Casein |----|
| C | u t | |----|
| H | i i | |----|
| E | - o | |----|
| M | n | |----|
| I +-----+----------------------+----+
| C | M | Acid curd |----|
| A | i | Rennet curd |----|
| L | l | Casein peptonized |----|
| | k | |----|
| F +-----+----------------------+----+
| E | Indol(3) |----|
| A | Hydrogen sulphide |----|
| T | Ammonia(3) |----|
| U | Nitrates reduced(3) |----|
| R | Fluorescent |----|
| E | Luminous |----|
| S | |----|
+---+----------------------------+----+
| D | Animal pathogen, epizoon |----|
| I | Plant pathogen, epiphyte |----|
| S | Soil |----|
| T | Milk |----|
| R | Fresh water |----|
| I | Salt water |----|
| B | Sewage |----|
| U | Iron bacterium |----|
| T | Sulphur bacterium |----|
| I | |----|
| O | |----|
| N | |----|
+---+----------------------------+----+
FOOTNOTES.
[1] Sir H. A. Blake has called attention to the fact that the “mosquito
theory” of malaria is mentioned in a Sanscrit manuscript of about the
6th century A.D.
[2] Myxomycetes excepted, and they are probably to be regarded as
animals--Mycetozoa.
[3] Centralblatt f. Bakteriologie, etc. LXIII. 1 Abt. Orig. 1912, 4,
idem LXVI. 1 Abt. Orig. 1912, 323.
[4] The pronunciation of this word according to English standards
is kok-si; the continental pronunciation is kok-kee; the commonest
American seems to be kok-ki. We prefer the latter since it is easier
and more natural and should like to see it adopted. (Author.)
[5] With the possible exception of blue green algæ which have been
found with bacteria in the above-mentioned hot springs. Seeds of
many plants have been subjected to as low temperatures as those
above-mentioned without apparent injury.
[6] It is popularly supposed that in canning fruit, vegetables, meats,
etc., all the air must be removed, since the organisms which cause
“spoiling” cannot grow in a vacuum. The existence of anaërobic and
facultative anaërobic bacteria shows the fallacy of such beliefs.
[7] “By cellulose is understood a carbohydrate of the general formula
C₆H₁₀O₅ not soluble in water, alcohol, ether, or dilute acids but
soluble in an ammoniacal solution of copper oxide. It gives with iodine
and sulphuric acid a blue color and with iodine zinc chloride a violet
and yields dextrose on hydrolysis.”--H. Fischer.
[8] The sulphur bacteria are partially prototrophic for S; probably the
iron bacteria also for Fe. Some few soil bacteria have been shown to
be capable of utilizing free H, and it seems certain that the bacteria
associated with the spontaneous heating of coal may oxidize free C. So
far as known no elements other than these six are directly available to
bacteria.
[9] Only a few kinds of bacteria so far as known are proto-autotrophic.
The nitrous and nitric organisms of Winogradsky which are so essential
in the soil, and which might have been the first of all organisms so
far as their food is concerned, and some of the sulphur bacteria are
examples.
[10] The term _pathogenic_ is also applied to certain non-parasitic
saprophytic bacteria whose products cause disease conditions, as one
of the organisms causing a type of food poisoning in man (_Clostridium
botulinum_), which also probably causes “forage poisoning” in domestic
animals.
[11] The term “fermentation” was originally used to denote the process
which goes on in fruit juices or grain extracts when alcohol and gas
are formed. Later it was extended to apply to the decomposition of
almost any organic substance. In recent years the attempt has been made
to give a chemical definition to the word by restricting its use to
those changes in which by virtue of a “wandering” or rearrangement of
the carbon atoms “new substances are formed which are not constitutents
of the original molecule.” It may be doubted whether this restriction
is justified or necessary. A definition is at present scarcely possible
except when the qualifying adjective is included as “alcoholic
fermentation,” “ammoniacal fermentation,” “lactic acid fermentation,”
etc.
[12] See “Oil and Gas in Ohio,” Bownocker: Geological Survey of Ohio,
Fourth Series, Bull. I, pp. 313-314.
[13] It is probable that this is the way “Jack o’lanterns” or “Will o’
the wisps” are ignited. Marsh gas is produced as above outlined from
the vegetable and animal matter decomposing in swampy places under
anaërobic conditions and likewise phosphine. These escape into the air
and the “spontaneous combustion” of the phosphine ignites the marsh gas.
[14] Dr. H. H. Green, of Pretoria, South Africa, has isolated from
“cattle dips” a bacterium that _reduces arsenates_ to _arsenites_.
[15] Dr. Green (l. c.) has also isolated an organism which causes some
deterioration of cattle dips by _oxidizing arsenites to arsenates_.
[16] It will be noted that the names of enzymes (except some of
those first discovered) terminate in _ase_ which is usually added to
the _stem of the name of the substance acted on_, though sometimes
to a word which indicates the substance formed by the action, as
_lactacidase_, _alcoholase_.
[17] Tetanus toxin is about 120 times as poisonous as strychnin, both
of which act on the same kind of nerve cells.
[18] In the author’s laboratory in the past ten years all sterilization
except those few objects in blood and serum work which must be dry, has
been done in autoclaves of the type shown in Fig. 81 which are supplied
with steam from the University central heating plant. A very great
saving of time is thus secured.
[19] The author has tested an “electric milk purifier” (Fig. 102)
which was as efficient as a first-class pasteurizer and left the milk
in excellent condition both chemically and as far as “cream line” was
concerned. The cost of operation as compared with steam will depend on
the price of electricity.
[20] The exact laboratory details for preparing various media are
not given in this chapter. It is the object to explain the choice of
different materials and the reasons for the various processes to which
they are subjected.
[21] For a discussion of this method of standardization consult the
following:
Clark & Lubs--J. Bact., 1917, II, 1-34, 109-136, 191-236.
Committee Report--Ibid., 1919. IV, 107-132.
Jones--J. Inf. Dis., 1919, 25, 262-268.
Fennel & Fisher--Ibid., 444-451.
Additional references will be found in these articles.
[22] Term also applied to the solidification of serum in media: _e.g._,
the Hiss inulin medium for the differentiation of pneumococci (see
diplococcus of pneumonia).
[23] The term “antigen” is also used to designate substances which may
take the place of what are supposed to be the true antigens in certain
diagnostic reactions (Chapter XXIX, Complement Fixation Test for
Syphilis).
[24] If the antitoxin is later concentrated (see last paragraph in
this chapter) a serum containing as little as 175 units per cc. may be
commercially profitable.
[25] Tho term “allergie” was introduced by Von Pirquet to designate the
state of the animal’s being sensitized and “allergic” as the adjective
derived therefrom. It does not seem to the author that there is any
advantage gained by the introduction of these terms.
INDEX
A
ABBÉ, 17
condenser, 200
microscope, improvements in, 30, 36
ABILGAARD, 26
Abrin, 262
Absorption of free nitrogen, 117
tests, 267
Accidental carriers, 241
structures, 43
Acetic acid, 99
bacteria, carbon oxidation, 114
fermentation, 32
_Acetobacter acidi oxalici_, 83
_xylinum_, 83
_Achorion schœnleinii_, 27, 34
Acid, acetic, 99
fermentation, 32
agglutination, 266
amino, relation to green plants, 119
butyric, 99
fermentation, 32, 99
carbolic, first used, 29
disinfectant action of, 159
fast bacteria, fat content, 84
staining of, 209
fermentation, 93
Bulgarian fermented milk, 98
ensilage, 98
industrial uses, 97
lactic acid, 96
sauerkraut, 98
hydrochloric, 246
production of, 110
soils, 81
Acquired immunity, 251, 252
_Actinomyces bovis_, 30, 36
Actinomycosis, cause of, 30, 36
path of entrance of, 244
Actions, reducing, 113
Activating enzymes, 125
Active immunity, definition of, 251, 252
production of, 252
Activities of bacteria, importance of, 31
overproduction, of cells, 258
physiological, definition of, 87
in identification, 216
Acute coryza, 244
disease, 233
Adulteration of food, anaphylactic test in, 293
complement-fixation test in, 279
immunity reactions in, 255
precipitin test in, 269
Aërobes, facultative, 76
strict, 76
Aërobic, 76, 215
Agar, composition of, 179
gelatinizing temperature, 179
medium, preparation of, 179
melting point of, 179
plating in, 188
sterilization of, 180
Agent, chemical, for disinfection, 156-163
choice of, for disinfection, 164
physical, for disinfection, 131
Agglutinating group, 266
Agglutination, acid, 266
diagnostic value of, 266
in identification of bacteria, 266
macroscopic, 265
microscopic, 265
phenomenon, 265
Agglutinin, 265
absorption test for, 267
action of, 266
anti-, 270
antigenic action of, 270
bacterial, 265
chief, 267
co-, 267
function of, 266
normal, 266
partial, 267
relation to precipitins, 269
specificity of, 267
theory of formation, 265
use of, 266
Agglutinogen, 266
Agglutinoid, 270
Aggressins, 288
Air, bacteria in, 71
filtration of, 153
“germ-free,” 153
Albumin in bacteria, 84
Alcohol as antiseptic, 160
as disinfectant, 160
Alcoholase, 125
Alcoholic fermentation, 31, 100
Alexin, 271, 273
Algæ, relation to bacteria, 37
Alimentary tract as path of entrance, 246
Alkalies as disinfectants, 158
Allergic, 290
Amboceptor, 273
anti-, 275
co-, 274
in cobra, 275
formation of, 273
hemolytic, 278
partial, 274
in rattle snake, 275
specificity of, 274
theory of formation, 273
Amboceptorogen, 274
Amebic dysentery, 29, 35
Ameboid cells, 247
colonies, 224
Amino-acids, relation to green plants, 119
Ammonia, structural formula, 103
Ammoniacal fermentation, 32
_Amœba coli_, 29, 35
Amphitrichic, 46
Amylase, 124
Anaërobes, 76
cultivation, methods of, 188
principles underlying, 188
facultative, 76
isolation of, 190
relation to elements, 86
strict, 76
Anaërobic, 76, 215
acid, butyric, 99
acid fermentation, 98
bacteria, first discovered, 32
fermentation of polysaccharides, 95
Analysis of ash, 82
chemical, of tubercle bacilli, 85
Anaphylactic, anti-, 290
phenomena, 292
reaction, uses of, 293
Anaphylatoxin, 290
Anaphylaxis, 289
anti-, 292
antibodies in, 291
theory of, 290, 291, 292
ANAXIMANDER, 18
ANDERSON, 289
ANDERSON and MCCLINTIC, phenol coefficient, 165
ANDRY, 25, 33
Anilin dyes, as antiseptic, 162
as disinfectants, 162
introduction of, 30
as stains, 204
Weigert, 36
fuchsin, 205
gentian violet, 205
water, 205
Animal carriers, 239
inoculation, uses of, 227
Animalcules, 19, 33
Animals, disinfection of, 170
experimental, 227
food relationships of, 39
_Ankylostoma duodenale_, discovery of, 27, 34
Egyptian chlorosis, cause of, 28, 35
hookworm disease, cause of, 28
Anthrax, 17, 28, 35
bacterium a facultative saprophyte, 238
isolation of, 29
due to a bacterium, 29
in human beings, 238
path of entrance, 243
intestine, 246
stomach, 246
persistence due to spores, 251
produced by exhaustion, 251
protective inoculation in, 30
spores, 29, 35
transmission by flies, 242
vaccine, 254
Anti-agglutinins, 270
aggressins, 288
amboceptors, 275
antisera in snake poisoning, 275
anaphylactic, 290
anaphylaxis due to intracellular digestion, 292
protein immunity compared to, 292
bacterial immunity, 254, 255
bodies, 259
place of production, 295
tabulation of, 294
body, action, 260
chemical composition, 260
formation of, 128, 260
complement, 274
complementophil amboceptor, 275
cytophil amboceptor, 275
diphtheritic serum, 263
enzyme, 122, 262
function of, 262
Antigen, 259
chemical composition of, 260
in complement-fixation, 277
syphilitic, 277, 279
in Wassermann test, 279
Antigens, fats and fatty acids as, 260
in preparation of vaccine, 285
tabulation of, 294
Antipollenin, 263
Antiprecipitins, 270
Antisepsis, 131
Lister, introduced, 35
primitive, 25
Antiseptic, 131
action of anilin dyes, 162
carbolic acid as, 159
cold as, 148
Antisera in snake poisoning, 275
Antisnake venoms, 275
Antitetanic serum, 263
Antitoxic immunity, 254, 255
Antitoxin, 261
collection of, 263
diphtheria, 30, 252
preparation of, 263
standard, 264
tetanus, 252
Antitoxins, 261-264
as factors in immunity, 295
preservative in, 263
specific, 261
Antivenin, 263
Apes, 227
Apparatus of Barber, 196
Appearance of growth on culture media, 217
APPERT, 20, 31, 34
Aqueous gentian violet, 205
Arborescent growth, 221
ARISTOTLE, 18
Aromatic compounds, production of, 104, 111
Arrak, 100
Arsenate, reduction of, 114
Arsenite, oxidation of, 115
ARTHUS, 289
phenomenon, 289
Articles, unwashable, disinfection of, 169
washable, disinfection of, 169
Artificial immunity, 251, 252
Ase, termination of name of enzyme, 124
Asepsis, 131
Aseptic, 131
Ash, analysis of, 82
Asiatic cholera, 27, 34, 73, 238, 239, 246, 248, 249
Attenuated, 253
Autoclave, air pressure sterilizer, 138
pressure sterilizer, 138
Autogenous vaccines, 284
in epidemic, 241
Autoinfection, 234
Autolysis, 149
self-digestion, 126
Autotrophic, 86
Available nitrogen, loss of, 113
Azotobacter, 118
B
BABES-ERNST corpuscles, 45
Bacilli, butter, 209
colon, 248
grass, 209
size and shape of, 52
tubercle, chemical analysis of, 85
Bacillus, 52, 60, 62
_anthracis_, 17, 36
spore staining, 209
Bacillus of blue milk, 31
Ducrey’s, 245
_subtilis_, 77, 83
spore staining, 209
Bacteria, absorption of N by, 117
acid fast, 84, 209
adaptability, range of, 90
advantage of motility to, 45
aids in isolation of, 197
anaërobic, 32
cause of disease in animals, 30
of souring of milk, 32
cell groupings of, 55
chains of, 38
chemical composition of, 39, 81
elements in, 82
classed as fungi, 37
as plants, 33, 35
definition of, 40
development of, 90
distribution of, 71
energy relationships, 39
environmental conditions for growth, 72
first classification of, 34
drawings of, 20
seen, 19, 33
food relationships of, 39
injurious, 72
isolation of, 194
measurement of, 40, 203
metabolism of, 86
methods of study of, 171
morphology of, 41
motile, 45
nitric, 114
nitrous, 114
nucleus of, 42
occurrence, 71
pathogenic, outside the body, 237
phosphorescent, 111, 112
position of, 37
rate of division, 43
of motion, 45
relation to algæ, 33, 37
to elements, 86
to gas and oil, 95
to phosphate rock, 115
to protozoa, 40
to soil fertility, 120
to sulphur deposits, 116
to yeasts and torulæ, 37
reproduction of, 37, 55
root tubercle, 86, 87
size of, 37, 40
soil, chief function of, 119
source of N, 102
speed of, 45
spiral, 53
staining of, 204-212
sulphur, 63
thermophil, 75, 77
universal distribution of, 90
in vinegar-making, 99
BACTERIACEÆ, 62, 66, 70
Bacterial agglutinin, 265
vaccines, 282
preparation of, 283, 284
Bacterin, 253
Bacteriocidin, 272
Bacteriological culture tubes, 184
examination, material for, 228
microscope, 200
Bacteriology, pathogenic, definition of, 231
reasons for study of, 217
as a science, 17, 32
Bacteriolysin, 272
Bacteriopurpurin, 62, 63, 112
Bacteriotropin, 281
_Bacterium abortus_, agglutinin of, 265
_coli_ in autoinfection, 234
gas formation by, 95
oxygen limits for, 77
pneumonia through intestinal route, 246
in preparation of sugar broths, 176
definition of, 62, 67, 70
_enteriditis_, cause of food poisoning, 104
_fluorescens_, oxygen limits, 77
_typhosum_, 73
agglutinin, 265
in phenol coefficient method, 166
pneumonia through intestinal route, 246
Ballon pipette, 193
Balsam, mounting in, 207
BARBER, 253
apparatus, 196
Barnyards, disinfection of, 167
Baskets, wire, 184
BASSI, 27
silkworm disease, 34
BASTIAN, 24
BAUMGÄRTNER, 256
Beaded growth, 221
Bed-bugs, 241
Beds, contact, 116
hot, 117
Beer, pasteurization of, 141, 144, 145
_Beggiatoa_, 63
BEGGIATOACEÆ, 63
BEHRING, 30
BELFANTI, 271
BERG, 27, 34
Berkefeld filter, 154
Bichloride of mercury as disinfectant, 158
BILHARZ, 28, 35
Bilharzia disease, 28, 35
Biochemical reactions, definition of, 87
Biological relationships, immunity reactions, 255, 270
Bipolar germination of spore, 48
Bismarck brown, 209, 212
Black-leg, 51, 73, 238, 243, 248, 251
vaccine, 254
Bleaching powder as disinfectant, 158
Blood, collection of, 228
cytolytic power of, 272
detection of, 269
serum, liquid, sterilization of, 182
Loeffler’s, 182
medium, preparation of, 182, 183
sterilization of, 182
vessels in dissemination of organisms, 247
Blue milk, bacterial cause of, 34
fermentation of, 31, 34
BOEHM, 27, 34
Boiling as disinfectant, 133
Boils, 237, 240, 243
BOLLINGER, 29, 30, 35, 36
BONNET, 20, 33
BORDET, 271
_Botrytis bassiana_, 27, 34
Bottles, staining of, 206
Bougies, 154
Bouillon, 173
BOYER, 260
Bread, salt rising, 95, 97
Bronchopneumonia, 233, 246
Broth, appearance of growth in, 218
extract of, 176
glycerine, 176
medium, 173
nitrate, 177
sterilization of, 174
sugar, 176
Brownian movement, 47, 203
Brushes, disinfection of, 169
Bubonic plague, 239
BUCHNER, 271
Budding of yeasts, 37
Bulgarian fermented milk, 98
Burning as disinfectant, 132
Burying as disinfectant, 154
BÜTSCHLI, 41, 43
Butter, 97
bacilli, staining of, 209
rancidity of, 101
Butyric acid fermentation, 32, 99
Buzzards, 241
C
CABBAGE disease due to protozoa, 36
Cadaverin, 104
CAIGNARD-LATOUR, 31, 34
Calcium hypochlorite as disinfectant, 158
oxide as disinfectant, 158
Candles, filter, 153, 154
Canned goods, food poisoning by, 104
spoilage of, 51
Canning, introduced, 21, 34
principles involved, 133
Capsule, 44, 45
of spore, 48
staining of, 210
Carbohydrates in bacterial cell, 84
fermentation of, 93-101
Carbol-fuchsin, 206
Carbolic acid as antiseptic, 159
as disinfectant, 159
first used, 29
Carbol-xylol, 209
Carbon cycle, 107
dioxide, 108
function of, in bacteria, 88, 101
oxidation of, 114
in proteins, liberation of, 105
source of, 88
uses of, 88, 101
CARBONI, 271
CARDANO, 18
Carrier problem, solution of, 240
Carriers, 239
accidental, 241
carrion eating animals as, 241
control of, 240
intermediate hosts as, 242
protective measures against, 242
universal, 240
of unknown organisms, 239
Cars, stock, disinfection of, 170
Catalase, 125
Catalytic agents, function of, 123
Catalyzer, 123
Cattle, 227
Causation of disease, 24, 128