DISINFECTION AND STERILIZATION (CONTINUED).
CHOICE OF AGENT.
The choice of the above-described agents depends on the conditions.
Evidently a barn is not to be disinfected in the same way that a
test-tube in the laboratory is sterilized. Among the factors to be
considered in making a choice are the thing to be disinfected or
sterilized, its size and nature, that is, whether it will be injured by
the process proposed, cost of the agent, especially when a large amount
of material is to be treated. Among the conditions which affect the
action of all agents the following should be borne in mind particularly
when testing the disinfecting power of chemical agents:
1. _The kind of bacterium_ to be destroyed, since some are more readily
killed by a given disinfectant than others, even though no spores are
present.
2. _The age of the culture._ Young bacteria less than twenty-four hours
old are usually more readily killed than older ones since the cell wall
is more delicate and more easily penetrated, though old growths may
be weakened by the accumulation of their products and be more easily
destroyed.
3. _Presence of spores_, since they are much more resistant than the
growing cells.
4. Whether the organism is a _“good” or “bad” growth_, _i.e._, whether
it has grown in a favorable environment and hence is vigorous, or under
unfavorable conditions and hence is weak.
5. _The number of bacteria present_, since with chemical agents the
action is one of relative masses.
6. _Nature of the substance in which the bacteria are._ Metallic salts,
especially bichloride of mercury, are precipitated by albuminous
substances and if employed at all must be used in several times the
ordinary strength. Solids require relatively more of a given solution
than liquids.
7. _State of the disinfectant_, whether solid, liquid or gas, and
whether it is ionized or not. Solutions penetrate best and are
therefore more quickly active and more efficient.
8. _The solvent._ Water is the best solvent to use. Strong alcohol (90
per cent. +) diminishes the effect of carbolic acid, formaldehyde and
bichloride of mercury. Oil has a similar effect. The action is probably
to prevent the penetration of the disinfectant.
9. _Strength of solution._ The stronger the solution, the more rapid
and more certain the action, for the same reason as mentioned under 5.
In fact, every disinfectant has a strength below the lethal at which it
stimulates bacterial growth.
10. _Addition of salts._ Common salt favors the action of bichloride
of mercury and also of carbolic acid. Other salts may hinder by
precipitating the disinfectant.
11. _Temperature._ Chemical disinfectants, as a rule, follow the
general law that chemical action increases with the temperature, up to
the point where the heat of itself is sufficient to kill.
12. _Time of action._ It is scarcely necessary to point out that a
certain length of time is necessary for any disinfectant to act. One
may touch a red hot stove and not be burned. All the above-mentioned
conditions are influenced by the time of action.
STANDARDIZATION OF DISINFECTANTS--“PHENOL COEFFICIENT.”
Many attempts have been made to devise standard methods for testing
the relative strengths of disinfectants. The one most widely used in
the United States is the so-called “Hygienic Laboratory” method of
determining the “phenol coefficient” of the given substance and is a
modification of the method originally proposed by Rideal and Walker
in England. In this method as proposed by Anderson and McClintic,
formerly of the above laboratory, the strengths of the dilution of the
disinfectant to be tested which kills a culture of _Bacterium typhosum_
in 2-1/2 minutes is divided by the strength of the dilution of carbolic
acid which does the same; and the dilution which kills in 15 minutes is
likewise divided by the corresponding dilution of carbolic acid. The
two ratios thus obtained are averaged and the result is the “phenol
coefficient.” For example
Phenol 1:80 killed in 2-1/2 minutes
Disinfectant “A” 1:375 „ „ „ „
Phenol 1:110 „ „ 15 „
Disinfectant “A” 1:650 „ „ „ „
375 ÷ 80 = 4.69
650 ÷ 110 = 5.91
-----
2)10.60
-----
Average = 5.30 = “phenol coefficient.”
Standard conditions of temperature, age of culture, medium, reaction,
etc., and of making the dilutions and transfers are insisted on.
Details may be found in the Journal of Infectious Diseases, 1911, 8, p.
1.
This is probably as good a method as any for arriving at the relative
strengths of disinfectants and in the hands of any given worker
concordant results in comparative tests can usually be attained.
Experience has shown that the results obtained by different workers
with the same disinfectant may be decidedly at variance. This is to
be expected from a knowledge of the factors affecting the action of
disinfectants above stated and from the known specific action of
certain disinfectants on certain organisms (compare anilin dyes, p.
162).
It seems that the only sure way to test the action of such a substance
is to try it out in the way it is to be used. It is scarcely wise to
adopt the “phenol coefficient” method as a legal standard method as
some states have done.
PRACTICAL STERILIZATION AND DISINFECTION.
The methods for sterilizing in the laboratory have been discussed and
will be referred to again in the next chapter.
In practical disinfection it is a good plan always to _proceed as
though spores were present_ even if the organism is known. Hence use an
_abundance of the agent_ and _apply it as long as practicable_. Also
it is best to secure the _chemical substances used as such_ and _not
depend on patented mixtures purporting to contain them_. As a rule the
latter are _more expensive_ in _proportion to the results secured_.
_Surgical instruments_ may be sterilized by boiling in water for
fifteen minutes, provided they are clean, as they should be. If dried
blood, pus, mucus, etc., are adherent, which should never be the case,
they should be boiled one-half hour. The addition of sodium carbonate
(0.5 to 1 per cent.) prevents rusting. Surgeons’ sterilizers are to
be had at reasonable prices and are very convenient. Whether the
instruments are boiled or subjected to streaming steam depends on
whether the supporting tray is covered with water or not. The author
finds it a good plan to keep the needles of hypodermic syringes in a
small wire basket in an _oil bath_. The oil may be heated to 150° to
200° and the needles sterilized in a very few minutes. The oil also
prevents rusting.
_Rooms_, _offices_ and all spaces which may be readily made practically
gas-tight are best disinfected by means of formaldehyde by any of the
methods above described (Figs. 111 and 112).
_Stables_ and _Barnyards_ (Mohler): “A preliminary cleaning up of all
litter is advisable together with the scraping of the floor, mangers
and walls of the stable with hoes and the removal of all dust and
filth. All this material should be burned since it probably contains
the infective agent. Heat may be applied to the surfaces, including
barnyard, by means of a ‘cyclone oil burner.’ When such burning is
impracticable, the walls may be disinfected with one of the following:
1. Whitewash 1 gallon + chloride of lime 6 ounces.
2. Whitewash 1 gallon + crude carbolic acid 7 ounces.
3. Whitewash 1 gallon + formalin 4 ounces.
The same may be applied with brushes or, more rapidly, sprayed on with
a pump; the surface soil of the yard and surroundings should be removed
to a depth of 5 or 6 inches, placed in a heap and thoroughly mixed
with quicklime. The fresh surface of soil thus exposed may be sprinkled
with a solution of a chemical disinfectant as above described.
[Illustration: FIG. 111.--Formaldehyde generator used in city work for
room disinfection.]
[Illustration: FIG. 112.--Government formaldehyde generator.]
“Portions of walls and ceiling not readily accessible may be
disinfected by chlorine gas liberated from chloride of lime by crude
carbolic acid. This is accomplished by making a cone of 5 or 6 pounds
of chloride of lime in the top of which a deep crater is made for the
placement of from 1 to 2 pints of crude carbolic acid. The edge of
the crater is thereupon pushed into the fluid, when a lively reaction
follows. Owing to the heat generated, it is advisable to place the
chloride of lime in an iron crucible (pot), and to have nothing
inflammable within a radius of two feet. The number and location of
these cones of chloride of lime depend on the size and structure
of the building to be disinfected. As a rule it may be stated that
chlorine gas liberated from the above sized cone will be sufficient for
disinfecting 5200 cubic feet of air space.”
_Liquid manure_, _leachings_, etc., where collected are thoroughly
disinfected by chloride of lime applied in the proportion of 2 parts to
1000 of fluid.
[Illustration: FIG. 113.--Chamber used in government work for
formaldehyde disinfection. The small cylinder at the side is the
generator.]
_Vehicles_ may be thoroughly washed with 2 per cent. formalin solution,
or if closed space is available, subjected to formaldehyde gas
disinfection, after cushions, hangings, etc., have been removed and
washed with the disinfectant.
_Harness_, _brushes_, _combs_ should be washed with a solution of
formalin, carbolic acid, or creolin as given under these topics.
_Washable articles_ should be boiled, dropped into disinfectant,
solutions as soon as soiled, and then boiled or steamed.
_Unwashable articles_--burn all possible. Use formaldehyde gas method
in a closed receptacle (Fig. 113).
_Stock cars_--the method described for stables is applicable here.
_Animals, large and small_, may have the coat and surface of the body
disinfected by washing with 1 to 1000 bichloride or strong hot soapsuds
to which carbolic acid has been added to make a 5 per cent. solution;
they should then be given a good warm bath.
Frequently time and money are saved by a combination of steam and
formaldehyde disinfection. This is a regular practice in municipal and
quarantine disinfection (Fig. 114).
[Illustration: FIG. 114.--Chamber in actual use at government
quarantine station for disinfecting baggage and dunnage with steam
or formaldehyde or both. The small cylinder at the side is the steam
formaldehyde generator.]
Persons engaged in disinfection work should wear rubber boots, coats
and caps which should be washed in a disinfectant solution and the
change to ordinary clothing made in a special room so that no infective
material will be taken away.
PART III.
THE STUDY OF BACTERIA.
CHAPTER XVI.
CULTURE MEDIA.
The study of bacteria may be taken up for the disciplinary and
pedagogic value of the study of a science; with the idea of extending
the limits of knowledge; or for the purpose of learning their
beneficial or injurious actions with the object of taking advantage of
the former and combating or preventing the latter.
Since bacteria are classed as plants, their successful study implies
their cultivation on a suitable soil. A growth of bacteria is called
a “_culture_” and the “soil” or material on which they are grown is
called a “_culture medium_.” In so far as the culture medium is made up
in the laboratory it is an “artificial culture medium” as distinguished
from a natural medium. A culture consisting of one kind of bacteria
only is spoken of as a “pure culture,” and accurate knowledge of
bacteria depends on obtaining them in “pure culture.” After getting
a “pure culture” the special characteristics of the organism must be
ascertained in order to distinguish it from others. The discussion of
the _morphology_ of bacteria in Chapters II, III, and IV shows that
the morphological structures are too few to separate individual kinds.
They serve at best to enable groups of similarly appearing forms to be
arranged. Hence any further differentiation must be based on a study
of the _physiology_ of the organism as discussed in the chapters on
Physiological Activities of Bacteria.
The thorough study of a bacterium involves, therefore:
1. Its isolation in pure culture.
2. Its study with the microscope to determine morphological features
and staining reactions.
3. Growth on culture media for determining its physiological activities
as well as morphological characteristics of the growths themselves.
4. Animal inoculations in certain instances.
5. Special serum reactions in some cases.
Since isolation in pure culture requires material for growing the
organism, the first subject to be considered is culture media.
A culture medium for a given bacterium should show the following
essentials:
1. It must contain all the elements necessary for the growth of the
organism except those that may be obtained from the surrounding
atmosphere.
2. These elements must be in a form available to the organism.
3. The medium must not be too dry, in order to furnish sufficient
moisture for growth and to prevent too great a concentration of the
different ingredients.
4. The reaction must be adjusted to suit the particular organism dealt
with.
5. There must be no injurious substances present in concentration
sufficient to inhibit the growth of the organism or to kill it.
Ordinarily, more attention must be paid to the sources of the two
elements N and C than to the others, for in general the substances
used to furnish these two and the water contain the other elements in
sufficient amount. For very exact work on the products of bacteria,
_synthetic media_ containing definite amounts of chemicals of known
composition have been prepared, but for most of the work with bacteria
pathogenic to animals such media are not needed.
Culture media may be either _liquid_ or _solid_, or for certain
purposes may be liquid at higher temperatures and solid at lower, as
indicated later. Liquid media are of value for obtaining bacteria for
the study of morphology and cell groupings and for ascertaining many of
the physiological activities of the organisms. Solid media are useful
for studying some few of the physiological activities and especially
for determining characteristic appearances of the isolated growths
of bacteria. These isolated growths of bacteria on solid media are
technically spoken of as “_colonies_,” whether they are microscopic in
size or visible to the unaided eye.
It is clear that the kinds of culture media used for the study of
bacteria may be unlimited but the undergraduate student will need to
use a relatively small number, which will be discussed in this section.
=Meat Broth (Bouillon).=[20]--This itself is used as a medium and as
the basis for the preparation of other solid and liquid media.
Finely ground _lean_ beef is selected because it contains the necessary
food materials. Fat is not desired since it is a poor food for most
bacteria and in the further processes of preparation would be melted
and form an undesirable film on the surface of the medium. The meat is
placed in a suitable container and mixed with about twice its weight of
_cold_ water (not distilled) and allowed to soak overnight or longer.
The cold water extracts from the meat water-soluble proteins, blood,
carbohydrates in the form of dextrose (occasionally some glycogen),
nitrogenous extractives and some of the mineral salts. The fluid is
strained or pressed free from the meat. This “meat juice” should now
be thoroughly boiled, which results in a coagulation of a large part
of the proteins and a precipitation of some of the mineral salts,
particularly phosphates of calcium and magnesium, both of which must be
filtered off and the water loss restored by adding the proper amount of
distilled water. The boiling is done at this point because the medium
must later be heated to sterilize it and it is best to get rid of the
coagulable proteins at once. The proteins thus thrown out deprive
the medium of valuable nitrogenous food material which is replaced
by adding about 1 per cent. by weight of commercial peptone. It is
usual also (though not always necessary) to add about 0.5 per cent. by
weight of common salt which helps to restore the proper concentration
of mineral ingredients lost by the boiling. The chlorine is also an
essential element. The reaction is now determined and adjusted to the
desired end point, “standardized,” as it is called. The medium is
again _thoroughly_ boiled and filtered boiling hot. The adjusting of
the reaction and the boiling ordinarily cause a precipitate to form
which is largely phosphates of the alkaline earths with some protein.
The filtered medium is collected in suitable containers, flasks or
tubes, which are plugged with well-fitting non-absorbent cotton plugs
and sterilized, best in the autoclave for twenty minutes at 15 pounds
pressure, or discontinuously in streaming steam at 100°. If careful
attention is paid to _titration_ and to _sufficient boiling_ where
indicated, the meat broth prepared as above should be clear, only
faintly yellowish in color and show no precipitate on cooling.
The conventional method for standardizing an acid medium is as follows:
Take 5 cc of the medium, add 45 cc of distilled water and 1 cc of
_phenolphthalein_ as indicator. Boil the solution and while still hot
run in from a burette N/20 NaOH solution until a faint pink color
appears. From the number of cc of N/20 NaOH used to “neutralize” the
5 cc of medium it is calculated how many cc of N/1 NaOH are necessary
to give the desired end reaction to the volume of medium which is to
be standardized. The resulting reaction is expressed as % _acid or
alkaline to phenolphthalein_. If it is necessary to add to each 100 cc
of the medium 1 cc of N/1 NaOH to make it neutral to phenolphthalein,
the reaction is called 1% acid: if to each 100 cc of medium there is
added 1 cc of N/1 alkali in addition to the quantity necessary to
neutralize, the reaction is called 1% alkaline.
In order to obtain a pink color when titrating with this indicator
not only must the “free acid” be neutralized by the alkali but also
loosely combined acid and any other substances present which will
combine with the alkali rather than with the indicator so that in many
media _more alkali_ is added than is necessary to neutralize the “free
acid,” _i.e._, the free H ions present.
It is well established that the controlling factor in the growth of
bacteria in so far as “reaction” is concerned is not the _titratable
substances_ present but only the “free acid,” _i.e._, the _number of
free H ions_, consequently it is better to determine the concentration
of H ions and to _standardize to a definite H ion concentration_.
Phenolphthalein as shown above is not a good indicator for this purpose.
The H ions present can be determined accurately in all cases only by
electrolytic methods. The apparatus necessary is usually relatively
expensive and scarcely adapted to the use of large classes of students.
There are a number of indicators each of which will show color changes
within rather narrow ranges of H ion concentration. Standardization by
the use of these indicators, the “colorimetric method,” is recommended
by the Society of American Bacteriologists and is coming into general
use.
The H ion concentration is ordinarily _indicated_ by the conventional
symbol P{H}, _e.g._, the concentration in pure water which is regarded
as neutral is expressed as P{H} 7; of normal HCl, P{H} 0; of normal
NaOH, P{H} 14. The figure after P{H} does not in reality represent the
concentration of H ions in the solution. This, like the concentration
of acids, is expressed on the basis of normality, _i.e._, as compared
with the concentration of a normal solution (1 g. equivalent) of H
ions. Concentration of H ions in pure water is N/10,000,000, _i.e._,
is 1/10,000,000 of the concentration in a normal solution of H ions.
Expressed in other words, it is the concentration in a normal solution
of H ions diluted ten million times. 10,000,000 = 10 to the 7th power
= 10⁷. Hence the figure after the P{H} indicates the _logarithm of
the number of times the solution is diluted_. Therefore this number
_increases with the dilution_, and the larger the figure after the
P{H}, the _less acid the solution is_.
Most saprophytic organisms and many parasitic ones grow within a wide
range of H ion concentration so that titration with phenolphthalein
gives sufficient accuracy for media for such organisms. On the
other hand, many organisms grow within a very narrow range of H ion
concentration, hence accurate standardization to a definite H ion
concentration is necessary. It is also evident that for comparative
work, such standardization is essential because this reaction can be
reproduced in other media and by other workers.[21]
Broth may be prepared from Liebig’s or Armour’s meat extract by adding
5 grams of either, 10 grams peptone and 5 grams NaCl to 1000 cc of
water, boiling to dissolve, then titrating and filtering as above.
The author after much experience finds _meat juice_ preferable to meat
extract for broth and other media for pathogenic bacteria, and has
abandoned the use of meat extracts for these organisms.
=Glycerin Broth.=--Glycerin broth is made by adding 4 to 6 per cent. of
glycerin to the broth just previous to the sterilization. The glycerin
serves as a source of carbon to certain bacteria which will not grow on
the ordinary broth--as _Mycobacterium tuberculosis_.
=Sugar Broths.=--Sugar broths are used for determining the action of
bacteria on these carbohydrates, since this is a valuable means of
differentiating certain forms, especially those from the intestinal
tract. Broth _free from sugar_ must first be made. This is done by
adding to broth prepared as already described, _just previous to final
filtering and sterilization_, a culture of some sugar-destroying
organism (_Bacterium coli_ is ordinarily used), and then allowing the
organism to grow in the raw broth at body temperature for twenty-four
hours. Any carbohydrate in the broth is destroyed by the _Bacterium
coli_. This mixture is then boiled to kill the _Bacterium coli_,
restandardized and then 1 per cent. by weight of required sugar is
added. Dextrose, saccharose and lactose are the most used, though
many others are used for special purposes. After the sugar is added
the medium must be sterilized by _discontinuous heating_ at 100° for
three or four successive days, because long boiling or heating in the
autoclave splits up the di- and polysaccharids into simpler sugars and
may even convert the simple sugars (dextrose) into acid.
Various other _modified broths_ are frequently used for special
purposes but need not be discussed here.
=Dunham’s peptone solution=, frequently used to determine indol
production, is a solution of 1 per cent. of peptone and 0.5 per cent.
of salt in tap water. It does not need to be titrated, but should be
boiled and filtered into tubes or flasks and sterilized.
=Nitrate Broth.=--Nitrate broth for determining nitrate reduction
is 1 per cent. of peptone, 0.2 per cent. of C. P. potassium nitrate
dissolved in distilled water and sterilized.
=Milk.=--Milk is a natural culture medium much used. It should be
fresh and thoroughly skimmed, best by a separator or centrifuge to
get rid of the _fat_. If the milk is not fresh, it should be titrated
as for broth and the reaction adjusted. The milk should be sterilized
discontinuously to avoid splitting up the lactose as well as action on
the casein and calcium phosphate.
_Litmus Milk._--Litmus milk is milk as above to which litmus has been
added as an acid production indicator. The milk should show blue when
the litmus is added or be made to by the addition of normal NaOH
solution. It should be sterilized discontinuously. Frequently on
heating litmus milk the blue color disappears due to a reduction of
the litmus. This blue color will reappear on shaking with air or on
standing several days, due to absorption of O and oxidation of the
reduced litmus, provided the heating has produced no other change in
the milk, as proper heating will not.
=Gelatin Culture Medium.=--Gelatin to the extent of 10 to 15 per
cent. is frequently added to broth and gives a culture medium of
many advantages. It is solid at temperatures up to about 25° and
fluid above this temperature, a property which is of great advantage
in the isolation of bacteria. (See Chapter XVIII.) Further gelatin
is liquefied (that is digested, converted into gelatin proteose and
gelatin peptone, which are soluble in water and do not gelatinize)
by many bacteria and not by others, a valuable diagnostic feature.
The gelatin colonies of many bacteria are very characteristic in
appearance, as is the growth of many on gelatin in culture tubes.
Gelatin medium may be prepared by adding the proper amount of gelatin
(10 to 15 per cent. by weight) broken into small pieces (powdered
gelatin in the same proportion may be used) to broth, gently warming
until the gelatin is dissolved, standardizing as for broth, filtering
and sterilizing. It is usually cleared before filtering by stirring
into the gelatin solution, cooled to below 60°, the white of an egg
for each 1000 cc., and then thoroughly boiling before filtering. The
coagulation of the egg albumen entangles the suspended matter so that
the gelatin filters perfectly clear, though with a slight yellowish
color. The filtering may be done through filter paper if the gelatin
is well boiled and filtered boiling hot, but is more conveniently done
through absorbent cotton, wet with boiling water.
Or, the gelatin may be added to _meat juice before it is boiled_, then
this is heated to about body temperature (not too hot, or the proteins
will be coagulated too soon) until the gelatin is dissolved. Then
the material is standardized and thoroughly boiled and filtered. The
proteins of the meat juice coagulate and thus clear the medium without
the addition of egg white. Commercial gelatin is markedly acid from the
method of manufacture, hence the medium requires careful titration,
even when made from a standardized broth.
Gelatin should be sterilized by discontinuous heating at 100° on three
successive days, because long boiling or heating above 100° tends to
hydrolyze the gelatin into gelatin proteose and peptone and it will
not gelatinize on cooling. It may be heated in the autoclave for
ten to fifteen minutes at 10 pounds’ pressure and sometimes not be
hydrolyzed, but the procedure is uncertain and very resistant spores
may not be killed. The medium should be put into the culture tubes in
which it is to be used as soon as filtered, and sterilized in these,
since, if put into flasks these must be sterilized, and then when
transferred to tubes for use, it must be again sterilized unless great
care is taken to have the tubes plugged and sterilized first, and in
transferring aseptically to these tubes. These repeated heatings are
very apt to decompose the gelatin, so it will not “set” on cooling. The
prepared and sterilized tubes of gelatin should be kept in an ice-box
or cool room, as they will melt in overheated laboratories in summer or
winter.
=Agar Medium.=--Agar agar, usually called agar, is a complex
carbohydrate substance of unknown composition obtained from certain
seaweeds along the coast of Japan and Southeastern Asia. It occurs
in commerce as thin translucent strips or as a powder. It resembles
gelatin only in the property its solutions have of gelatinizing when
cooled. Gelatin is an albuminoid closely related to the proteins,
agar a carbohydrate. Agar is much less soluble in water, 1 or 1.5
per cent. of agar giving a jelly as dense as 10 to 15 per cent. of
gelatin. It dissolves only in water heated to near the boiling-point
(98° to 99°) and only after much longer heating. This hot solution
“jells,” “sets” or gelatinizes at about 38° and remains solid until
again heated to near boiling. Hence bacteria may be grown on agar at
the body temperature (37°) and above, and the agar will remain solid,
while gelatin media are fluid above about 25°. No pathogenic bacteria
and none of the saprophytes liable to be met with in the laboratory are
able to “liquefy” agar.
An agar medium is conveniently prepared from broth by adding 1 or
1.5 per cent. of the finely divided agar to the broth and boiling
until dissolved, standardizing, clearing, filtering, and sterilizing.
The agar must be thoroughly boiled, usually for ten to fifteen
minutes, and the water loss made up by the addition of distilled
water before titration. Agar is practically neutral so that there is
little difference between the titration of the dissolved agar and
the original broth. The agar solution should be kept hot from the
beginning to the end except the cooling down to below 60°, when the
egg white for clearing is added. Though filtration through paper is
possible as with gelatin, if the agar solution is thoroughly boiled
and filtered boiling hot, it is more satisfactory for beginners to
use absorbent cotton wet with boiling water and to pour the hot agar
through the same filter if not clear the first time. The solidified
agar medium is never perfectly clear, but always more or less
opalescent. The agar medium may be sterilized in the autoclave for
fifteen minutes at 15 pounds pressure as the high temperature does not
injure the agar.
=Potato Media.=--Potatoes furnish a natural culture medium which is
very useful for the study of many bacteria. The simplest, and for most
purposes the best, way to use potatoes is in culture tubes as “potato
tube cultures” (No. 8, Fig. 119). These are prepared as follows:
Large tubes are used. Large healthy potatoes are selected. Each end
of the potato is sliced off so as to have parallel surfaces. With a
cork-borer of a size to fit the tubes used, cylinders about one and
one-half inches long are made. Each cylinder is cut diagonally from
base to base. This furnishes two pieces each with a circular base and
an oval, sloping surface. The pieces are then washed clean and dropped
for a minute into boiling water to destroy the oxidizing enzyme on the
surface which would otherwise cause a darkening of the potato. (The
darkening may also be prevented by keeping the freshly cut potatoes
covered with clean water until ready to sterilize.) A bit of cotton
one-fourth to one-half inch in depth is put into each of the test-tubes
to retain moisture and a piece of potato dropped in, circular base
down. The tubes are then plugged with cotton and sterilized in the
autoclave at 15 pounds pressure for not less than twenty-five minutes,
since potatoes usually harbor very resistant spores, and it is not
unusual for a few tubes to spoil even after this thorough heating.
Potatoes are sometimes used in “potato plate cultures.” The term “plate
culture” is a relic of the time when flat glass plates were used for
this and other “plate cultures.” Now glass dishes of the general
form shown in Fig. 115, called “Petri dishes,” or plates are used for
practically all plate culture work. For “potato plates” slices from
potatoes are cut as large and as thick as the relative sizes of potato
and dish permit (Fig. 116). The slices should be thin enough not to
touch the lid and thick enough to be firm.
[Illustration: FIG. 115.--Petri dish with the lid partly raised. × 1/2]
[Illustration: FIG. 116.--A potato plate. × 1/2]
It is a good plan to wrap each dish separately in paper to retain the
lid securely, then sterilize as for potato tubes, and leave plates
wrapped until wanted.
It sometimes happens that the natural acidity of potatoes is too great
for the growth of many organisms. The acidity is sufficiently corrected
by soaking the pieces of potato in a 1 per cent. solution of sodium
carbonate for an hour before they are put into the tubes or plates.
_Glycerinized potato tubes_ are conveniently prepared by covering the
potato in the tube with glycerin broth, sterilizing and pouring off the
excess broth immediately after sterilizing, taking care that the tubes
do not become contaminated which is not very probable if the work is
quickly done while the tubes are still hot.
=Blood Serum Media.=--Blood serum, usually from the larger, domestic
animals on account of convenience in securing it in quantity, is used
in the study of the bacteria causing disease in man and animals. Most
commonly the serum is collected from the clotted blood after it has
well separated (usually about forty-eight hours is required for this).
It is then run into tubes which are plugged with cotton and placed in
an apparatus for coagulating the serum by heat. A copper water bath
with a tightly closed air compartment or the horizontal autoclave (Fig.
81) is sufficient for this purpose, though special forms of apparatus
are to be had. It is important that the temperature be raised slowly so
that the blood gases escape gradually. Three to five hours or longer
should be allowed for the temperature to reach the boiling-point. If
the tubes are heated too rapidly, the serum is filled with bubbles and
badly torn since the gases are driven off suddenly. _Löffler’s serum_
is made by adding one part of dextrose broth to three parts of serum
and then coagulating as above. The solidified serum in either case is
best sterilized discontinuously, though with care the autoclave at 15
pounds pressure may be used for a single sterilization. This is very
apt to cause a greater darkening of the serum and frequently also a
laceration of the solid mass by escaping gases.
Blood serum is also used in the liquid state. For this purpose it is
best to collect it aseptically; or it may be sterilized discontinuously
at a temperature of 55° or 56° on seven to ten consecutive days. Novy
has recently suggested dialyzing the serum to free it from salts and
thus prevent its coagulation when heated. Whether the removal of the
various “extractives” which diffuse out with the salts deprives the
serum of any of its advantageous properties remains to be ascertained.
From the discussion of the physiological activities of bacteria in
Chapters IX-XII it is apparent that a very great variety of culture
media other than those described is necessary for the study of special
types of bacteria, but such media are beyond the scope of the present
work.
The ideal culture media are without a doubt the _synthetic media_, that
is media of definite known chemical composition, so that the various
changes due to the growth of bacteria can be accurately determined
and thus a means of sharply differentiating closely related organisms
be secured. Such media have been prepared and every bacteriologist
believes strongly in their future usefulness when media of wider
application shall have been devised. An example of this type of culture
media is Uschinsky’s synthetic medium, of which the following is one of
the modifications:
Distilled-water 1000 parts
Asparagin 4 „
Ammonium lactate 6 „
Disodium phosphate 2 „
Sodium chloride 5 „
A criticism of this medium is that the elements K, Ca, Mg, Fe, Mn, and
S which have been shown to be essential are not present if chemically
pure salts are used in the preparation.
CHAPTER XVII.
METHODS OF USING CULTURE MEDIA.
The way in which culture media shall be used depends on the purpose
in view. By far the larger part of bacteriological work is done with
cultures in “bacteriological culture tubes.” Various laboratories have
their own special types but all are more or less after the “Board of
Health” form. They differ from ordinary chemical test-tubes in that
they are usually longer, have no “lip” and have much thicker walls to
prevent breakage and consequent loss of the culture as well as danger
from pathogenic organisms. The author finds two sets of tubes most
serviceable for student use--one size 15 cm. long by 19 mm. outside
diameter (No. 9, Fig. 119), the other 15 cm. long by 13 mm. (Nos. 1 to
7, Fig. 119). Culture tubes are conveniently used in “wire baskets”
circular or square in section and of a size to correspond with the
length and number of tubes used. These baskets are light, do not break,
and if made of good galvanized wire netting do not readily rust (Figs.
117 and 118).
Liquid media such as broth, milk, litmus milk, indol and nitrate broths
are used in the above-mentioned tubes when small quantities only are
to be worked with. The tubes are filled approximately one-third full,
then plugged with _non-absorbent_ cotton and sterilized. _Cotton plugs_
are used so much in bacteriological work because they permit a free
circulation of air and gases and at the same time act as filters to
keep out the bacteria of the air.
Sugar broths or other media in which gas may be produced are used in
fermentation tubes (Smith tubes) of the type shown in Fig. 120 so that
the gas may be collected in the closed arm of the tube, measured (Fig.
121) and tested if desired.
[Illustration: FIG. 117.--Round wire basket.]
[Illustration: FIG. 118.--Square wire basket.]
[Illustration: FIG. 119.--Culture tubes with media in them. × 2/3. _1_
to _7_ are the smaller tubes mentioned in the text; _9_ the larger
tube; _8_ is extra large for potato tubes; _1_, plain broth; _2_, plain
milk; _3_, litmus milk; _4_, gelatin for “stab” or “puncture” culture;
_5_, agar for “stab” or “puncture” culture; _6_, agar for “slope” or
“slant” culture; _7_, blood serum; _8_, potato tube; _9_, agar for
plating. Note the transparency of the broth and gelatin and the slight
opalescence of the agar.]
One method of using gelatin and also agar is as “puncture” or “stab”
cultures. The tubes (the narrower tubes are to be preferred for most
“stab” cultures) are filled one-third full of the medium while it is
still fluid, plugged, sterilized and allowed to cool in the vertical
position. The medium is then “inoculated” with a _straight_ platinum
needle by plunging this into the center of the surface down to the
bottom of the tube (Fig. 119, Nos. 4 and 5).
[Illustration: FIG. 120.--Fermentation tubes. _1_, filled ready for
use; _2_, shows a cloudy growth and the development of gas in the
closed arm.]
Agar and blood serum are frequently used in the form of “slope” or
“slant” cultures. That is, the medium solidifies with the tubes lying
on their sides which gives a long, sloping _surface_ on which the
bacteria are inoculated (Fig. 119, Nos. 6 and 7).
[Illustration: FIG. 121.--Method of estimating percentage of gas in a
fermentation tube by means of the “gasometer”, the reading is 45 per
cent.]
[Illustration: FIG. 122.--A toxin flask showing a large surface
growth.]
Potato tubes are likewise used for “slant” or “slope” cultures (Fig.
119, No. 8). Potatoes as “plate cultures” have been referred to. Agar
and gelatin are very largely used in the form of “plate cultures”
also. For this purpose Petri dishes are first sterilized, then the
melted agar or gelatin poured into them and allowed to “set” while the
plates are kept horizontal. The melted media may be “inoculated” before
they are poured, or a portion of the material to be “plated” may be
placed in the dish, then the melted medium poured in and distributed
over the dish by tilting in various directions, or the medium after
solidifying may be inoculated by “strokes” or “streaks” over its
surface, according to the purpose in view in using the plate. The
larger sized tubes should be used for making plates in order to have
sufficient medium in the plate (No. 9, Fig. 119).
For using large quantities of medium, Florence flasks, Ehrlenmeyer
flasks, special toxin flasks (Fig. 122) or various other devices
(Vaughan and Novy’s “mass cultures,” Figs. 123 and 124) have been
employed.
For growing _anaërobic organisms_ it is evident that some method for
removing and excluding the oxygen of the air must be used. A very great
variety of appliances have been devised for these purposes. Some are
based on the principle of the vacuum, exhausting the air with an air
pump; some on replacing the air with a stream of hydrogen; others on
absorbing the oxygen by chemical means, as with an alkaline solution
of pyrogallic acid, or even by growing a vigorous aërobe in the
same culture or in the same container with the anaërobe, the aërobe
exhausting the oxygen so that the anaërobe then develops, or finally
by excluding the air through the use of deep culture tubes well filled
with the medium, or in the closed arm of fermentation tubes. For many
purposes a combination of two or more of the above methods gives good
results.
In any event the culture medium should have been _freshly sterilized_
just before use, or _should be boiled_ in order to drive out the
dissolved oxygen. For most, anaërobes the presence in the medium of
about 1 per cent. of a carbohydrate, as dextrose, is advisable.
A description of all the various devices is unnecessary in this work,
but the following have answered most of the purposes of general work in
the author’s laboratories.
[Illustration: FIG. 123.--Tank with raised lids. (Vaughan.)]
[Illustration: FIG. 124.--Tank with lids lowered. (Vaughan.)
FIGS. 123 and 124.--Vaughan and Novy’s mass culture apparatus.]
_A._ “_Vignal tubes_” of the style shown (Fig. 125) are made from
glass tubes of about 6 to 8 mm. outside diameter, sealed at the small
end, plugged with cotton above the constriction and sterilized. The
medium, agar or gelatin, which has been previously inoculated with the
anaërobic culture, is then drawn up into the tube, after breaking off
the tip, as far as the constriction. The tube is then sealed in the
flame at the small end and also at the constriction. Since it is full
of the medium and sealed, access of air is prevented. This forms an
excellent means for “isolation” (Chapter XVIII); the tube needs merely
to be cut with a file at the point where colonies appear, then these
may be readily transferred.
[Illustration: FIG. 125.--Vignal tubes. × 1/3 _1_, the sterile tube
ready for inoculation; _2_, fourth dilution tube showing a few isolated
colonies, one near the figure; _3_, third dilution showing colonies
isolated but numerous; _4_, second dilution tube showing colonies still
more numerous; _5_, first dilution tube showing colonies so numerous
and small as to give a cloudy appearance to the tube. In use tube _2_
would be filed in two at the colony and inoculations made from it.]
_B._ “_Fermentation tubes_” form a simple means for growing liquid
cultures of anaërobes, the growth occurring in the closed arm only,
while with facultative anaërobes, growth occurs both in the closed arm
and in the open bulb. A little “paraffin oil” (a clear, heavy petroleum
derivative) may be poured on the fluid in the open bulb as a very
efficient seal, though it is not usually necessary.
_C._ “_Deep culture tubes._”--The medium, agar, gelatin or a liquid is
poured into tubes until they are approximately one-half full, a little
paraffin oil is poured on the surface (not essential always), then the
tubes are plugged and sterilized. Inoculation is made to the bottom
and anaërobes grow well (Fig. 126).
[Illustration: FIG. 126.--Deep tubes showing anaërobic growth. _1_,
shows a few small gas bubbles; _2_, shows the medium broken up by the
excessive development of gas.]
_D._ For slope or plate, or any type of surface cultures the Novy jar
(Fig. 127) is the most practical device. It is good practice to combine
the vacuum method, the hydrogen replacement method and the oxygen
absorption method in using these jars. In operation a solution of 20
per cent. NaOH is poured on the bottom of the jar to a depth of 1 or 2
cm., the cultures are placed on glass supports above the alkali and a
short wide tube of strong pyrogallol is set in on the bottom in such a
way that it may be easily upset and mixed with the alkali when it is
desired to do so. The cover is now clamped in position with all joints
well vaselined. Then the outlet tube is connected with a suction pump
and the air drawn out. Meanwhile the inlet tube has been connected
with a hydrogen generator, and after the jar is exhausted hydrogen is
allowed to flow in, and this process is repeated until one is satisfied
that the air is replaced. The suction exhausts the air from the tubes
or plates so that much less time is required to replace the air with
hydrogen. Finally the stop-cock is closed, and the pyrogallol solution
is gently shaken down and mixed with the alkali so that any remaining
oxygen will be absorbed.
[Illustration: FIG. 127.--Novy jars.]
It must be remembered that facultative anaërobes as well as anaërobes
will grow under any of the above conditions, so that cultures of
organisms so obtained must be further tested aërobically in order to
determine to which group the organisms belong.
Reference has been made above to the “inoculation” of culture media,
which means introducing into the medium used the desired material in
the proper way. For small quantities this is most conveniently done
with platinum “needles,” that is, pieces of platinum wire inserted
into the ends of glass rods. The “straight” needle is a piece of
heavy platinum wire of about 0.022 inch in diameter (Fig. 128). It is
used most frequently to inoculate all forms of _solid media_. The
platinum loop is of lighter wire, 0.018 inch. The loop in the end is
conveniently made by twisting the wire around the lead of an ordinary
lead-pencil. The “loop needle” (Fig. 129) is most used in transferring
liquid media. On account of the high price of platinum, the author
has substituted “nichrome” wire for student use. This is stiffer, not
so easily made into loops and breaks out of the rods more easily.
The latter defect is remedied to some extent, by imbedding the wire
only slightly for about one-fourth of an inch on the side of the end
portion of the rod. The low cost, less than one-twentieth of platinum,
justifies its use.
[Illustration: FIG. 128.--Straight needle.]
[Illustration: FIG. 129.--Straight and loop needles.]
[Illustration: FIG. 130.--Pasteur flask--“ballon pipette.”]
Sterile graduated pipettes varying in capacity from 1 cc graduated in
hundredths, upward, permit the transfer of definite amounts of liquids.
Large quantities are conveniently transferred by means of Pasteur
flasks (Fig. 130). The details of inoculation are best derived from
laboratory practice.
CHAPTER XVIII.
ISOLATION OF BACTERIA IN PURE CULTURE.
As has been stated, the thorough study of a bacterium depends on
first getting it in pure culture. In the early days of bacteriology
supposedly pure cultures were obtained by (1) _dilution in liquid
media_. A series of tubes or flasks containing sterile liquid media was
prepared. Number one was inoculated with the material to be examined
and thoroughly mixed. A small portion of the mixture was transferred
to number two, and mixed; from this to number three, and so on until a
sufficient number were inoculated, the last three or four in the series
receiving the same amounts of a very high dilution of the original
material. If one or two of these latter showed a growth and the others
not, it was assumed that the dilution had been carried so far that only
a single organism was transferred and therefore the culture obtained
was “pure.” The method in this crude form is too uncertain to be of
value today and recourse is had to more exact means. The procedure most
widely used is that of (2) “_plating out_” by means of gelatin or agar
plates. The material to be plated out is diluted by transferring to
three or more tubes of melted gelatin or agar as in the first method
and then all the tubes are poured into Petri dishes and grown under
suitable conditions. By proper mixing in the tubes the bacteria are
well scattered through the medium which holds the individual organisms
separate when it solidifies. On some of the plates a sufficient
dilution will be reached so that the colonies developing from the
bacteria will be so few that they are separate and pure cultures may
be obtained by inoculating from one of these a tube of the appropriate
medium (Figs. 131 to 134). The chief uncertainty with this method is
that occasionally two kinds of bacteria stick together so closely that
even the separate colonies contain both organisms. This is not common,
however. The plate colonies frequently develop from groups of bacteria
which were not separated, but as these are of the same kind the culture
is essentially pure.
[Illustration: FIG. 131.--Dilution plates. × 3/10. _1_, shows the
first dilution, the colonies are so numerous and small that they
are invisible (compare Fig. 132); _2_, shows fewer and hence larger
colonies, but too crowded to isolate (compare Fig. 133); _3_, shows the
colonies larger and well separated, so that it is easy to isolate from
them (compare Fig. 134).]
[Illustration: FIG. 132.--A portion of plate _1_ in Fig. 131 as seen
under the low-power objective. × 100. Very small, closely crowded
colonies.]
Another method which is frequently applicable with material from human
or animal sources is to (3) _rub the material over the surface_ of a
slope tube or of medium solidified in a Petri dish with a sterile heavy
platinum needle, glass rod, or cotton swab. If the bacteria are not
too numerous, pure cultures may frequently be obtained. A modification
of this method is to make a series of (4) _parallel streaks on a
slope tube or plate of medium_ with a needle inserted _but once_ into
the material to be plated. On the first streak most of the bacteria
are rubbed off and a continuous growth results, but usually on the
last of a series only isolated colonies appear, which are presumably
pure. The ideal method for securing pure cultures is to be absolutely
certain that the culture starts from a single organism. This may be
accomplished by means of the (5) _apparatus and pipettes devised by
Professor Barber_ of the University of Kansas (Figs. 135 and 136). With
this instrument a single organism is picked out under the microscope
and isolated in a drop of culture medium and observed until it is
seen to divide, thus proving its viability. Transfers are then made
to the proper media. The method requires much practice to develop the
necessary skill in the making of pipettes, determining the proper
condition of the large cover-glasses used over the isolating box, and
in manipulation, but the results fully compensate.
[Illustration: FIG. 133.--From the thinnest part of plate _2_, Fig. 131
as seen under the low-power objective. × 100. Colonies much larger than
on plate _1_, but still crowded.]
[Illustration: FIG. 134.--The smallest colony on plate _3_, Fig. 131,
as seen under the low-power objective. × 100. Large, single, isolated
colony.]
Professor W. A. Starin of the author’s department, a former student of
Professor Barber, has done some excellent work with this apparatus.
[Illustration: FIG. 135.--Diagram of Barber’s isolation apparatus. _b_,
moist chamber; _ms_, large cover-glass over moist chamber; _p_, small
pipette drawn out to a fine point; _k_, _r_, _g_, pipette holder; _f_,
screw for raising and lowering _k_, _r_, _g_; _s_, screw for lateral
motion of _k_, _r_, _g_; _n_, screw for clamp on pipette which allows
it to be moved in or out; _m_, mechanical stage of microscope; _t_,
rubber tube held in the mouth and used to move the liquid culture
medium in the pipette. (Journal of Infectious Diseases, October 20,
1908, vol. 5, No. 4, p. 381.)]
[Illustration: FIG. 136.--Photograph of microscope with Barber’s
isolation apparatus set up to use.]
A number of procedures may be used to greatly facilitate the above
methods of isolation by taking advantage of the different physiological
properties of different organisms in a mixture such as ability
to form spores, different resistance to antiseptics, special food
requirements, and pathogenic properties. (_a_) If material contains
resistant spores, it may be _heated to temperatures high_ enough to
kill all of the organisms except the spores (80° for half an hour, for
example) and then plated out. Or (_b_) _an antiseptic which restrains
the growth_ of some organisms and not others may be placed in the
culture media (carbolic acid, various anilin dyes, (p. 162), excess
acid, or alkali, ox bile, etc.), when the more resistant organisms grow
on the final plates, the others not. (_c_) _Special food substances_
(various carbohydrates) from which the organism desired forms special
products (acids, aldehydes) that may be shown on the plates by various
indicators, is one of the commonest means. Or media in which certain
organisms thrive and others not, so that the former soon “crowd out”
the latter (unsterilized milk for lactic acid bacteria, inorganic
media in soil bacteriology) may be used. A combination of the general
methods (_b_) and (_c_) is much used in the separation of the organisms
of the “intestinal group” in human practice. (_d_) _The inoculation
of a susceptible animal_ with a mixture suspected to contain a given
pathogenic bacterium frequently results in the development of the
latter in pure culture in the body of an animal, from which it may be
readily recovered. In all of the above methods (except Barber’s) the
first “pure culture” obtained should be “purified” by replating in a
series of dilution plates to make sure that it is pure.
CHAPTER XIX.
STUDY OF INDIVIDUAL BACTERIA--STAINING.
When an organism has been obtained in pure culture by any of the
methods described in the preceding chapter the next step is the study
of its morphology as discussed in Chapters II--IV. This involves the
use of the microscope, and since bacteria are so small, objectives
of higher power than the student has presumably used will be needed.
Doubtless only the two-thirds inch or 16 mm. and the one-sixth inch or
4 mm. objectives are all that have been used in previous microscopic
work, while for examining bacteria a one-twelfth inch or 2 mm. is
necessary. It will have been observed that the higher the power of
the objective the smaller is the front lens or object glass and
consequently the less is the amount of light which enters. With the use
of the one-twelfth inch or 2 mm. objective it is necessary to employ
two devices for increasing the amount of light entering it, with which
the student is probably not familiar. One of these is to place a drop
of cedar oil between the front lens and the object and to immerse the
lens in this oil--hence the term “oil-immersion objective;” the other
is the substage or Abbé condenser. The latter is a system of lenses
placed below the stage and so constructed as to bring parallel rays of
light--daylight--from an area much larger than the face of the front
lens of the objective to a focus on the object to be examined, thus
adding very greatly to the amount of light entering the objective.
Since the condenser brings _parallel_ rays to a focus on the object,
the _flat-mirror_ is always used with the condenser when working with
daylight. With _artificial light close_ to the microscope, the concave
mirror may be used to make the divergent rays more nearly parallel and
thus give better illumination.
The function of immersion oil is to prevent the dispersion of
considerable light that would otherwise occur owing to refraction
as the light passes up through the slide and into the air. The
accompanying diagram will help to make this clearer (Fig. 137). A ray
of light (_A B_) coming through the slide will be refracted in the
direction _B C_ if the medium has a lower refractive index than the
slide, as air has, and hence will not enter the objective _O_. If,
however, there is interposed between the objective and the slide a
medium which has the same refractive index as the slide, as immersion
oil has, then the ray will continue in the same direction (_B D_) at
the point _B_ and hence enter the objective. Evidently the immersion
oil causes much more light to enter the front lens and makes the field
brighter and at the same time prevents considerable refraction and
dispersion of light from the object seen and hence this appears more
distinct and sharply defined. The Abbé condenser and the oil-immersion
objective are practically always used in the microscopic study of
bacteria (Fig. 138).
[Illustration: FIG. 137.--Diagram of use of immersion oil.]
[Illustration: FIG. 138.--Diagram of paths of rays of microscope.]
HANGING DROP SLIDE.
It is sometimes necessary to examine living bacteria and for this
purpose the device known as the “hanging drop slide” is used (Fig.
139). The slide has a slight concave depression ground in the middle
of one face. A ring of vaseline is placed around this depression with
the loop needle. On a clean cover-glass, large enough to fit over the
ring of vaseline, several drops of a broth culture, or of material
from a solid culture suspended in broth or physiological normal salt
solution are placed. The slide is inverted on the cover-glass in such
a way that the ring of vaseline seals the latter to the slide. When
the whole preparation is quickly turned cover side up, the drops are
seen “hanging” to the under side of the cover over the depression in
the slide. In examining such a preparation with the microscope great
care is necessary in order to focus on the bacteria, without breaking
the cover. To see the organisms distinctly the _lower iris diaphragm of
the condenser must be nearly closed_, so that the light coming through
consists mainly of parallel vertical rays, otherwise the transparent
bacteria themselves refract and diffract the light and appear blurred
and indistinct. By studying living bacteria with this device it can
be determined whether they are motile or not. The motility should not
be confounded with the familiar “Brownian movement” of all minute
insoluble inert particles which non-motile living bacteria and
also dead bacteria show. The hanging drop slide is of value in the
measurement of bacteria, since this is properly done on the living
organism. Measurement is done with a calibrated ocular micrometer as in
other kinds of measurement with the microscope with which the student
is presumably familiar. The direct effect of various agents on living
bacteria as light, electricity, heat, etc., in the study of “tropisms”
and “taxes” has been investigated on various modifications of the
above-described hanging drop slide.
[Illustration: FIG. 139.--Hanging drop slide.]
Cell forms and cell groupings may be studied in the same way but
these features are best determined on _stained_ preparations in many
instances.
“Dark field” illumination and the ultramicroscope are of great value in
the study of living bacteria and other minute objects, but apparatus
of this type would scarcely be used by the student in an introductory
course, so that they will not be discussed in the present volume.
STAINING.
The main use of the microscope in bacteriology is in the study of
_stained preparations_ of the organisms. Staining makes bacteria opaque
and hence more easily seen than the transparent unstained forms. Some
methods of staining also show morphological structures which are either
imperfectly recognized in the unstained cell, spores, or are not
visible at all--capsules, metachromatic granules, flagella. Finally
certain bacteria are colored by special methods of staining which do
not affect others, so that under proper conditions these bacteria may
be recognized by staining methods alone--tubercle bacilli in the organs
of animals.
The phenomena of staining are essentially chemical, though sometimes
the chemical union is a very weak one, even resembling an absorption of
the dye rather than true chemical union--most watery stains. In other
cases the chemical compounds formed are decidedly stable and are not
decomposed even by strong mineral acids--staining of tubercle bacilli
and other “acid-fast” organisms. In still other cases the principal
action is a precipitation on the surface of the object stained--methods
for staining flagella.
In many methods of staining in addition to the dyes used other
substances are added to the solution which assist in fixing the dye in
or on the organism stained. Such substances are called _mordants_. The
principal mordants used are alkalies, anilin, carbolic acid, iodine,
metallic salts, tannic acid.
While it is true that some bacteria may be stained by that standard
histological nuclear dye, hematoxylin, it is of little value for this
purpose. Practically all bacteriological stains are solutions of the
_anilin dyes_. These dyes, as is well known, are of nearly every
conceivable color and shade but relatively very few are used in
bacteriological work. The beginning student will rarely use solutions
of other than the three dyes _fuchsin_ (red), _methylene blue_ and
_gentian violet_ for staining bacteria, with occasionally Bismarck
brown, or eosin, or safranin as tissue contrast stains.
The bacteriological dyes are kept “in stock” as saturated solutions in
95 per cent. alcohol which are _never used as stains_, but merely for
convenience in making the various staining solutions.
The approximate percentages of the three common dyes in such solutions
are indicated in the following table adapted from Woods _Chemical and
Microscopical Diagnosis_, Third Edition, 1917, Appendix:
Fuchsin 3.0%
Gentian Violet 4.8%
Methylene Blue 2.0%
The stains made from these dyes which are in most common use are the
following:
1. Aqueous (watery) gentian violet solution.
Saturated alcoholic solution of gentian violet 1 part
Distilled water 20 parts
Mix well and filter.
2. Anilin gentian violet.
Saturated alcoholic solution of gentian violet 1 part
Anilin water (see below) 10 parts
Mix well and filter.
3. Anilin Fuchsin.
Saturated alcoholic solution of fuchsin 1 part
Anilin water (see below) 10 parts
Mix and filter.
These stains rarely keep longer than ten days in the laboratory (unless
kept in the ice-box) and must be made fresh on the first sign of a
deposit on the glass of the container.
=Anilin Water.=--Anilin water is made by putting 3 or 4 cc of anilin
“oil” in a 120 cc. flask, adding 100 cc of distilled water, shaking
vigorously for a minute or so and filtering through a wet filter, in
other words, a saturated solution of anilin in water.
4. Löffler’s (methylene) blue.
Saturated alcoholic solution of methylene blue 3 parts
Aqueous solution of NaOH (or KOH), 1 to 10,000 10 „
Mix and filter.
5. Carbol-fuchsin (Ziehl’s solution).
Saturated alcoholic solution of fuchsin 1 part
5 per cent. aqueous solution of carbolic acid 10 parts
Mix and filter.
6. Gabbet’s (methylene) blue (solution).
Dry methylene blue 4 parts
Concentrated H₂SO₄ 25 „
Distilled water 75 „
Dissolve the dry dye in the acid and add the solution to the
distilled water and filter.
[Illustration: FIG. 140.--Author’s staining set. Square bottles are set
in square holes in the block. The capacity of each bottle is 30 cc.]
Staining solutions are conveniently kept in square dropping bottles
inserted in a block as shown in Fig. 140. This form of holder
necessitates the use of _one hand only_ in securing the stain and
dropping it on the preparation.
The actual staining of bacteriological preparations can be learned only
by repeated laboratory practice, yet the following methods have given
such uniform results in class work that it is felt they are not out of
place in a text-book.
=Preparation of the “Film.”=--The author learned to stain bacteria,
on the “cover-glass” but does not recall having used this method in
fifteen years and does not teach it to his students. All staining is
done on the slide. To prepare a film from a solid culture medium the
procedure is as follows:
First, be sure the slide is clean and _free from grease_. This is
accomplished most readily by scouring a few minutes with finely
ground pumice stone and a little water, then washing and drying with
a grease-free cloth, handkerchief, or piece of cheese-cloth. With the
“loop” needle place in the middle of the slide a small loop of water.
This is best done by filling the loop by dipping in water, then tapping
it gently so that all that remains is the water that just fills the
loop level full, and this amount is placed on the slide by touching
the flat side of the loop to the glass. Then the _straight needle_ is
sterilized, dipped into the culture and just touched once into the
small drop of water on the slide. The remainder of the culture on the
straight needle is then burned off and the needle is used to spread the
drop of water containing the bacteria into a thin even film, which will
result, provided the slide is free from grease. This is dried and then
“fixed” by passing three times through the Bunsen flame at intervals of
about one second, passing through slowly for thick slides and a little
more rapidly for thin ones. If the culture is in a liquid medium, the
use of the loop of water is unnecessary; a loop of the fluid from the
surface, middle or bottom as the culture indicates is spread out to a
thin film, dried and fixed.
After the film is fixed the stain desired is dropped on, allowed
to act for the proper time, which will depend on the stain and the
preparation, washed in water, dried thoroughly and examined with the
oil-immersion lens, without a cover. If it is desired to preserve the
preparation it may then be mounted in balsam. This is not necessary, as
they keep just as well, provided the immersion oil is removed. To do
this, fold a piece of filter paper so that at least three thicknesses
result. Lay this on the slide and press firmly several times, when the
surplus oil will be taken up by the paper. Slides not mounted in balsam
are more apt to become dusty than those that are. This is the only
disadvantage.
=Gram’s Method of Staining.=--It has been ascertained that some
bacteria contain a substance, possibly a protein, which forms a
compound with gentian violet and iodine, which compound is insoluble in
alcohol, and other bacteria do not contain this substance. Consequently
when bacteria are stained by Gram’s method (given below), those that
contain this chemical remain colored, while if it is not present the
dye is washed out by the alcohol and the bacteria are colorless and may
be stained by a contrast stain. The bacteria which stain by this method
are said to “take Gram’s” or to be “Gram-positive,” while those that
decolorize are called “Gram-negative.” The method is:
1. Prepare the film as above given.
2. Stain with fresh anilin gentian violet 1 minute.
3. Wash in tap water.
4. Cover with Gram’s solution 1 minute.
5. Wash in tap water.
6. Wash with 95 per cent. alcohol three times or until no more color
comes out.
7. Dry and examine.
Gram’s solution is:
I 1 part
KI 2 parts
H₂O 300 „
This method is excellent for differentiating Gram-positive and
Gram-negative organisms on the same slide. First stain by this
method and after washing with alcohol stain with a counter-stain,
carbol-fuchsin diluted ten to fifteen times with water is excellent.
The Gram-positive bacteria are violet and the Gram-negative are red.
It is also of great value in staining Gram-positive bacteria in
tissues, but the sections should be stained about five minutes in
the anilin gentian violet and be left about two minutes in the Gram’s
solution. Sections are to be counter-stained in Bismarck brown, dilute
eosin or safranin solutions and cleared in oil of bergamot, lavender or
origanum and not in clove oil or carbol-xylol, as these latter dissolve
out the dye from the bacteria.
=Staining of Spores in the Rod.=--Prepare the films as usual. Cover
with carbol-fuchsin, using plenty of stain so that it will not dry on
the slide; heat until vapor arises, not to boiling; cool until the
stain becomes cloudy and heat again until the stain clears, and repeat
once more; wash in tap water and then wash in 1 per cent. H₂SO₄ three
times, dropping on plenty of acid, tilting and running this over the
slide three times and then pour off and use fresh acid and repeat this
once. Wash thoroughly in _distilled_ water, then stain with Löffler’s
blue one to three minutes. Wash, dry and examine. The spores should be
bright red in a blue rod.
This method will give good results if care is taken to secure cultures
of the right age. If the culture is too old the spores will all be free
outside the rods, while if too young they will decolorize with the
acid. For _Bacillus subtilis_ and _Bacillus anthracis_, cultures on
agar slants forty-eight hours in the 37° incubator are just right. For
the spores of _Clostridium tetani_, the culture should be three days
old, but may be as old as a week.
=Staining of “Acid-fast” Bacilli.=--_Mycobacterium tuberculosis,
Mycobacterium of Johne’s disease, “grass” and “butter bacilli,”
Mycobacterium lepræ, Mycobacterium smegmatis._
_Gabbet’s method_:
1. Prepare the film as usual.
2. Stain with carbol-fuchsin as given above for spores.
3. Wash with tap water.
4. Decolorize and stain at the same time with Gabbet’s blue, two or
three minutes.
5. Wash, dry and examine.
The sulphuric acid in Gabbet’s blue removes the carbol-fuchsin from
everything except the “acid-fast” bacteria, which remain red, and the
blue stains the decolorized bacteria and nuclei of any tissue cells
present.
_Ziehl-Neelson method_:
1, 2, 3, as in Gabbet’s method.
4. Decolorize with 10 per cent. HCl until washing with water shows
only a faint pink color left on slide.
5. Wash thoroughly.
6. Stain with Löffler’s blue one or two minutes.
7. Wash, dry and examine.
The results are the same as with Gabbet’s method.
=Staining of Capsules.=--_Räbiger’s Method._--Films of the organism
to show capsules should be _freshly prepared, dried but not fixed_.
Material is usually obtained from milk or blood. A drop of the fluid
is placed on the middle of a slide about one-fourth of the distance
from one end. The narrow edge of another clean slide is placed in this
drop and then drawn lengthwise across the slide with firm pressure.
This gives a _thin layer_ which is necessary if good results are to
be expected. The preparation is covered with a _freshly prepared_
saturated solution of gentian violet in formalin and this allowed
to stain for 30 seconds. Then wash _lightly_, dry and examine. The
organisms appear deeply violet and much larger than with ordinary
stains and capsules are well stained and show well.
_Welch’s Method._--Prepare films as in the above method. Cover with
glacial acetic acid for 10 to 20 seconds. Wash off the acid with
carbol-fuchsin. Wash the stain off with physiological normal salt
solution (0.85 per cent.) until all surplus stain is removed. Dry and
examine. Capsules and bacteria are red.
=Staining of Flagella.=--The rendering of flagella visible is
considered one of the most difficult processes in staining. Experience
of a number of years during which whole classes numbering from one
hundred to three hundred students accomplish this result shows that it
is no more difficult than many other staining processes. The essentials
are: (1) clean slides, (2) young cultures on agar slopes, (3) freshly
prepared mordant and stain which are kept free from precipitate,
(4) gentle heating. The author’s students are furnished only stock
materials and make their own cultures, mordants and stains.
The slides are cleaned with pumice in the usual way. An agar slope
culture of the organism to be stained from six to twenty-four hours
old is selected. A bit of the culture is removed and placed in a
watch-glass of water. The bacteria are allowed to diffuse of themselves
without stirring. After several minutes a loop of this water is removed
and three streaks are made across the slide, one in the middle and one
on each side of this about one-quarter of an inch from it. This gives
well scattered bacteria in one of the three streaks at least and very
little other material on the slide to cause precipitates. The slide is
carefully dried and fixed and then covered with an abundance of the
mordant by filtering through a small filter onto the slide so that the
mordant shows transparent on the slide. The preparation is then gently
warmed and cooled three times, adding mordant if necessary. _Do not
heat to steaming._ After mordanting for about five minutes the excess
is washed off under the tap. It is a good plan to hold the slide level
and allow the water to run into the center of the mordant and flow it
off. Inclining the slide is apt to cause the film on the surface of the
mordant to settle down on the slide and spoil the preparation. After
the mordant is washed off and all traces of it removed with a clean
cloth if necessary the stain is applied and gently heated and cooled
the same way for from three to five minutes. The preparation is then
washed, dried and examined.
The mordant used is a modification of Löffler’s which is somewhat
simpler in preparation since the stock solution of FeCl₃ is more
permanent than FeSO₄ solution.
Mordant sufficient for one student:
5 per cent. solution of FeCl₃ 20.0 cc
25 per cent. solution of tannic acid 20.0 cc
Anilin fuchsin 4.0 cc
Normal NaOH 1.5 cc
The solution of FeCl₃ is made up in the cold and must be perfectly
clear. The tannic acid solution must be thoroughly boiled and filtered
until clear. The iron and the acid are carefully mixed, boiled and
filtered clear. The anilin fuchsin must be added slowly with constant
stirring and the mixture boiled and filtered. The NaOH is added in the
same way and this mixture boiled and filtered. The final mordant should
not leave a film on a clean slide when poured on and allowed to run
off. Unless the mordant is in this condition and perfectly clear, it
should not be used, but a new one must be made up. Time and care in the
preparation of the mordant are essential.
The stain to follow this mordant is anilin fuchsin.
=Staining of Metachromatic Granules.=--_Neisser’s Method._ Prepare the
film in the usual way. Stain with Neisser’s stain a few seconds only.
Wash and stain with Bismarck brown a few seconds only.
_Neisser’s Stain_:
Sat. alcoholic solution of methylene blue 1.0 part
Glacial acetic acid 2.5 parts
Distilled water 50.0 parts
_Bismarck Brown_:
Bismarck brown (dry dye) 2 parts
Distilled Water 1000 parts
By the use of the hanging drop slide and the methods of staining just
described all the various morphological features of the bacterial cell
may be ascertained.
It is necessary when _cell groupings_ as characteristic of definite
modes of division are to be determined to make slides from a liquid
culture, as broth. Place a drop of the material, preferably from the
bottom of the tube in most instances, from the top in case a pellicle
or scum is formed on the surface, on the slide and allow this to dry
_without spreading it out_, fix, wash gently with water, then stain
lightly with Löffler’s blue. Such slides also show characteristic
_cell forms_ as well. Slides should be made from solid media to show
variations in form and size and involution forms. These latter are
especially apt to occur on potato media.
CHAPTER XX.
STUDY OF THE PHYSIOLOGY OF BACTERIA.
Of the environmental conditions influencing the growth of bacteria the
following are the chief ones ordinarily determined:
_A._ Temperature.--The optimum temperature for growth is usually
about the temperature of the natural environment and ordinarily one
determines merely whether the organism grows at body temperature (37°)
and at room temperature (20°) or not. For exact work the maximum,
minimum and optimum temperature must be ascertained by growing in
“incubators” with varying temperatures.
A bacteriological incubator is an apparatus for growing bacteria at a
constant temperature. This may be any temperature within the limits for
bacterial growth. If temperatures above that of an ordinary room are
desired, some source of artificial heat is needed. Electricity, gas or
oil may be used. A necessary adjunct is some device for maintaining
the temperature constant, a “thermoregulator” or “thermostat.” For
lower temperatures a cooling arrangement must be installed. For the
great part of bacteriological work only two temperatures are used, 20°
so-called “room temperature” (this applies to European “rooms” not to
American) and 37° or body temperature. Incubators for 37° of almost any
size and style desired may be secured from supply houses and need not
be further described. Figs. 141 and 142 illustrate some of the types.
For use with large classes “incubator rooms” are to be preferred. The
author has one such room for 37° work with 200 compartments for student
use which did not cost over $60 to install.
[Illustration: FIG. 141.--Small laboratory incubator, gas heated.]
[Illustration: FIG. 142.--Electric incubator.]
The styles of incubators for lower temperatures, 20° and below, are not
so numerous nor so satisfactory. The author has constructed a device
which answers every purpose for a small class. The diagram, Fig. 143,
explains it.
[Illustration: FIG. 143.--Diagram of fittings for a cold incubator.
_1._ small tank for constant head, about 1 foot in each dimension. _a_,
inflow; _b_, overflow; _c_, lead pipe. _2_, refrigerator. _a′_, ice;
_b′_, flat coil under ice; _c′_, outflow to incubator. _3_, incubator.
_a″_, cold water inflow; _b″_, overflow; thermometer and burner
omitted. The diagram explains the construction. The water cooled to
about 14° with artificial ice by flowing through the lead coil under
the ice, flows into the incubator which may be heated and regulated in
the usual way.]
The thermal death-point is determined by exposing the organisms in
thin tubes of broth at varying temperatures for ten-minute periods and
then plating out to determine growth. The effect of heat may also be
determined by exposing at a given temperature, _e.g._, 60°, for varying
lengths of time and plating out.
_B._ Oxygen relations--whether the organism is aërobic, anaërobic, or
facultative is determined by inoculation in gelatin or agar puncture or
stab cultures and noting whether the most abundant growth is at the
top, the bottom or all along the line of inoculation.
_C._ Reaction of the medium--acid, alkaline or neutral as influencing
the rate and amount of growth.
_D._ The kind of medium on which the organism grows best.
_E._ The effect of injurious chemicals, as various disinfectants, on
the growth.
_F._ Osmotic pressure conditions, though modifying decidedly the growth
of bacteria, are not usually studied as aids in their recognition, nor
are the effects of various forms of energy, such as light, electricity,
_x_-rays, etc.
Among the “Physiological Activities” discussed in Chapters IX-XII those
which, in addition to the staining reactions described, are of most
use in the identification of non-pathogenic bacteria are the first ten
listed below. For pathogenic bacteria the entire thirteen are needed.
1. Liquefaction of gelatin.
2. Digestion of blood serum.
3. Coagulation and digestion of milk.
4. Acid or gaseous fermentation in milk, or both.
5. Acid or gaseous fermentation of various carbohydrates in
carbohydrate broth, or both.
6. Production of indol in “indol solution.”
7. Production of pigments on various media.
8. Reduction of nitrates to nitrites, ammonia, or free nitrogen.
9. Production of enzymes as illustrated in the above activities.
10. Appearance of growth on different culture media.
11. Production of free toxins as determined by injection of animals
with broth cultures filtered free from bacteria.
12. Causation of disease as ascertained by the injection of animals
with the bacteria themselves, and recovery of the organism from the
animals.
13. Formation of specific antibodies as determined by the
proper injection of animals with the organism or its products
and the subsequent testing of the blood serum of the inoculated
animals.
For special kinds of bacteria other activities must be determined
(oxidation, nitrate and nitrite formation, action of sulphur and iron
bacteria, etc.).
The first nine activities are determined by inoculating the different
culture media already described and observing the phenomena indicated,
making chemical tests where necessary.
APPEARANCE OF GROWTH ON DIFFERENT CULTURE MEDIA.
In addition to those changes that are associated with the manifestation
of different physiological activities, many bacteria, show
characteristic appearances on the various culture media which are of
value in their identification.
Too much stress should not be laid on these appearances alone, however,
since slight variations, particularly in solid media due especially to
the age of the medium, may change decidedly the appearance of a colony.
This is true of variations in the amount of moisture on agar plates.
Colonies which are ordinarily round and regular may assume very diverse
shapes, if there chance to be an excess of moisture on the surface.
Also in slope and puncture cultures on the various solid media much
variation results from the amount of material on the inoculation needle
and just how the puncture is made, or the needle drawn over the slope.
These variations are largely prevented by the use of standard media and
by inoculating by standard methods. The Laboratory Committee of the
American Public Health Association has proposed standard methods for
all culture media and tests and for methods of inoculation, and these
have been generally adopted in this country for comparative work.
Likewise the Society of American Bacteriologists has at different times
(1904, 1914, 1917) adopted “descriptive charts” for detailing all the
characteristics of a given organism. A committee is at present working
on a revision of the 1917 chart to be presented at the 1920 meeting.
One of the earlier charts which includes a glossary of descriptive
terms is inserted in this chapter.
Among the cultural appearances the following are of most importance:
[Illustration: FIG. 144.--Broth cultures × 2/3. _1_ uninoculated
transparent broth; _2_, broth cloudy from growth of organisms; _3_,
broth slightly cloudy with a deposit in bottom; _4_, broth slightly
cloudy with a heavy membrane at the surface.]
[Illustration: FIG. 145.--A filiform stab or puncture culture. × 3/5.]
[Illustration: FIG. 146.--A beaded stab or puncture culture. × 1/2.]
[Illustration: FIG. 147.--A villous stab or puncture culture. × 1/2.]
In broth cultures the presence or absence of growth on the surface
and the amount of the same. Whether the broth is rendered cloudy or
remains clear, and whether there is a deposit at the bottom or not
(Fig. 144). An abundant surface growth with little or nothing below
indicates a strict aërobe, while a growth or deposit at bottom and a
clear or nearly clear medium above, an anaërobe. These appearances are
for the first few days only of growth. If the broth is disturbed, or
after the culture stands for several days many surface growths tend to
sink to the bottom. So an actively motile organism causes in general
a cloudiness, especially if the organism is a facultative anaërobe,
which tends to clear up by precipitation after several days when the
organisms lose their motility. Non-motile facultative anaërobes
usually cloud the broth also, but settle out more rapidly than the
motile ones.
In gelatin and agar punctures the oxygen relationship is shown by
surface growth for aërobes, growth near the bottom of the puncture
for anaërobes, and a fairly uniform growth all along the line of
inoculation for facultative anaërobes. In the case of these last
organisms, a preference for more or less oxygen is indicated by the
approach to the aërobic or anaërobic type of growth.
[Illustration: FIG. 148 FIG. 149 FIG. 150 FIG. 151
FIG. 148.--Crateriform liquefaction of gelatin. × 1/2.
FIG. 149.--Funnelform liquefaction of gelatin. × 1/2.
FIG. 150.--Saccate liquefaction of gelatin. × 1/2.
FIG. 151.--Stratiform liquefaction of gelatin. × 1/2.]
Along the line of puncture the commonest types are _filiform_ (Fig.
145), which indicates a uniform growth; _beaded_ (Fig. 146), or small
separate colonies; _villous_ (Fig. 147), delicate lateral outgrowths
which do not branch; _arborescent_, tree-like growths branching
laterally from the line. In agar these branchings are usually short and
stubby, or technically, _papillate_.
[Illustration: FIG. 152.--Filiform slope culture. × 1/2.]
[Illustration: FIG. 153.--Filiform, slightly spreading, slope culture.
× 1/2.]
[Illustration: FIG. 154.--Beaded slope culture. × 1/2.]
Further, in the gelatin puncture the liquefaction which occurs is
frequently characteristic. It may be _crateriform_ (Fig. 148), a
shallow saucer at the surface; or _funnel-shaped_ (Fig. 149); or it
may be of uniform width all along the puncture, _i.e._, _saccate_ (Fig.
150); or it may be _stratiform_, (Fig. 151), _i.e._, the liquefaction
extends to the sides of the tube and proceeds uniformly downward.
[Illustration: FIG. 155 FIG. 156 FIG. 157 FIG. 158
FIG. 155.--Effuse slope culture. × 1/2.
FIG. 156.--Rhizoid slope culture. × 1/2.
FIG. 157.--Rugose slope culture. × 1/2.
FIG. 158.--Verrucose slope culture. × 1/2.]
On agar, potato and blood serum slope tubes the amount of growth, its
form and elevation, the character of the surface, and the consistency
should be carefully noted, and in some few cases the character of the
edge. Figures 152 to 158 show some of the commoner types.
[Illustration: FIG. 159.--Punctiform colonies on a plate. × 1/2.]
[Illustration: FIG. 160.--A rhizoid colony on a plate. Natural size.]
[Illustration: FIG. 161.--Ameboid colonies on a plate. × 1/2.]
[Illustration: FIG. 162.--Large effuse colony on a plate. The edge is
lacerated. Incidentally the colony shows the rate of growth for six
successive days. × 2/3.]
[Illustration: FIG. 163.--Colony with edge entire as seen under the
low-power objective. × 100.]
[Illustration: FIG. 164.--Colony with edge coarsely granular as seen
under the low-power objective. × 100.]
[Illustration: FIG. 165.--Colony with edge curled as seen under the
low-power objective. × 100.]
[Illustration: FIG. 166.--Colony with edge rhizoid as seen under the
low-power objective. × 100.]
[Illustration: FIG. 167.--A small deep rhizoid colony as seen under the
low-power objective. × 100.]
On agar and gelatin plates made so that the colonies are well isolated,
the form of the latter, the rate of their growth, the character of the
edge and of the surface, the elevation and the internal structure
as determined by a low-power lens are often of almost diagnostic
value. Also in the case of the gelatin plates, the character of the
liquefaction is important. Figs. 159 to 167 show some of the commoner
characteristics to be noted.
[Illustration: FIG. 168.--A small mold colony natural size as viewed by
transmitted light.]
[Illustration: FIG. 169.--The same colony as viewed by reflected light.]
[Illustration: FIG. 170.--A portion of the thin edge of the same colony
as seen with the lower-power objective. × 100.]
[Illustration: FIG. 171.--A single fruiting body (sporangium) from the
same colony as seen under the lower-power objective. × 100.]
Colonies of mold frequently appear on plates. These are readily
differentiated from bacterial colonies after a little experience. With
the naked eye usually the fine radiations of the edge of the colony
are apparent. The surface appears duller and by reflected light more
or less “fuzzy.” With the low-power objective the relatively large,
branching threads of the mold (mycelia) show distinctly. Also the large
fruiting bodies (sporangia) are easily distinguished. Figs. 168 to 171
illustrate a common black mold (_Rhizopus nigricans_).
CHAPTER XXI.
ANIMAL INOCULATION.
Animal inoculation has been referred to (1) as a method of assisting
in the preparation of pure cultures of pathogenic organisms; (2) as a
means of testing the poisonous properties of substances produced in
bacterial cultures; (3) in order to test the ability of an organism to
cause a disease; (4) for the production of various antibodies; it may
be added (5) that some bacteria produce in the smaller experimental
animals lesions which do not occur in animals naturally infected, but
which nevertheless are characteristic for the given organism. The best
illustration is the testicular reaction of young male guinea-pigs to
intraperitoneal injections of glanders bacilli. Experimental animals
are also inoculated (6) to test the potency of various bacterial and
other biological products, as toxins, antitoxins, etc.
Guinea-pigs are the most widely used experimental animals because they
are easily kept and are susceptible to so many diseases on artificial
inoculation. Rabbits are used very largely also, as are white mice. For
special purposes white rats, pigeons, goats and swine are necessary.
For commercial products horses (antitoxins) and cattle (smallpox
vaccine) are employed. In the study of many human diseases the higher
monkeys and even the anthropoid apes are necessary, since none of the
lower animals are susceptible.
The commonest method of animal inoculation is undoubtedly the
_subcutaneous_. This is accomplished most readily with the hypodermic
needle. The skin at the point selected (usually in guinea-pigs the
lateral posterior half of the abdominal surface, in mice the back near
the root of the tail) is pinched up to avoid entering the muscles and
the needle quickly inserted. Clipping the hairs and washing with an
antiseptic solution should precede the inoculation as routine practice.
Frequently a small “skin pocket” is all that is needed. The hair is
clipped off, the skin pinched up with small forceps and a slight snip
with sharp scissors is made. The material may be inserted into this
pocket with a heavy platinum needle. _Cutaneous_ inoculation is made
by shaving the skin and rubbing the material onto the shaved surface
or scratching with a scalpel or special scarifier, but without drawing
blood, and then rubbing in the material to be inoculated.
_Intravenous_ injections are made with larger animals. In rabbits the
posterior external auricular is a convenient vein. In larger animals
the external jugular is used.
_Intraperitoneal_, _-thoracic_, _-cardiac_, _-ocular_, _-muscular_
injections, and injections into the parenchyma of internal organs are
accomplished with the hypodermic needle. In the case of the first two,
injury to contained organs should be carefully avoided. Intracardiac
injection, or aspiration of the heart to secure blood, requires
considerable practice to be successful without causing the death of the
animal at once through internal hemorrhage. In _subdural_ injections
into the cranial cavity it is necessary to trephine the skull first,
while such injections into the spinal canal may be accomplished between
the vertebra with needles longer and stronger than the usual hypodermic
needle. Occasionally animals are caused to _inhale_ the organisms, or
are _fed_ cultures mixed with the feed.
SECURING AND TRANSPORTING MATERIAL FROM ANIMALS FOR BACTERIOLOGICAL
EXAMINATION.
If the site of the lesion is readily accessible from the exterior,
material from the _living animal_ should be collected with sterile
instruments and kept in sterile utensils until the necessary tests can
be made. Testing should be done on material as soon after collection
as possible, in all cases, to avoid the effects of “decomposition”
bacteria.
If the blood is to be investigated it may be aspirated from a
peripheral vein with a sterile hypodermic syringe of appropriate size
or allowed to flow through a sterile canula into sterile receptacles.
The site of the puncture should be shaved and disinfected before the
instrument is introduced.
Discharges of whatever kind should likewise be collected in sterile
receptacles and examined as soon as may be.
If internal organs are to be examined it is best to kill a moribund
animal than to wait for death, since after death, and in severe
infections even sometimes before, the tissues are rapidly invaded by
saprophytic bacteria from the alimentary and respiratory tracts which
complicate greatly the isolation of the specific organism. Hence the
search for specific bacteria in carcasses or organs several hours after
death is frequently negative. Animal inoculation with such material is
very often followed by sepsis or septicemia in a few hours, so that the
specific organism has no opportunity to manifest itself.
In securing material for cultures from internal organs it is a good
plan to burn the surface of the organ with a gas or alcohol flame, or
to sear it with a hot instrument to kill surface organisms, then make
the incision or puncture through the burned area and secure material
from the interior of the organ. Such punctures made with a stiff
platinum needle frequently give pure cultures of the organism sought.
Slides may be made from such material and culture media inoculated at
once.
Since a bacteriological diagnosis depends most commonly on growing the
organisms, it is evident that material sent for examination must _never
be treated with an antiseptic or preservative_. If decomposition is to
be feared the only safe procedure is to _pack the material in ice_ and
forward in this way.
_Tuberculous material from the parenchyma_ of internal organs may be
forwarded in a preservative (not _formalin_, since this makes it very
difficult to stain the bacteria) as _in this special_ case a very
positive diagnosis may be made by staining alone. Even here it is
better to _pack in ice_ in order that the diagnosis by staining may be
confirmed by inoculating the living organisms into guinea-pigs.
In the case of material _from a rabid animal_ and many protozoal
diseases the rule against preservatives is not absolute, since staining
is a reliable diagnostic means. Even in these cases it is often
desirable to inoculate animals, hence, as before stated, it is best to
make it a uniform practice to _pack material for examination in ice and
use no preservatives_.
PART IV.
GENERAL PATHOGENIC BACTERIOLOGY.
CHAPTER XXII.
INTRODUCTION.
Pathogenic Bacteriology treats of the unicellular microörganisms which
are responsible for disease conditions, _i.e._, pathological changes
in other organisms. Hence not only are bacteria considered, but also
other low vegetable forms, as yeasts and molds, likewise protozoa in
so far as they may be pathogenic. For this reason the term pathogenic
“Microbiology” has been introduced to include all these organisms. It
is largely for the reason that the methods devised for the study of
bacteria have been applied to the investigation of other microörganisms
that the term “bacteriology” was extended to cover the entire field.
The general discussion in this chapter is intended to include,
therefore, microörganisms of whatever kind pathogenic to animals.
The term pathogenic as applied to an organism must be understood in a
purely _relative_ sense, since there is no single organism that can
cause disease in all of a certain class, but each is limited to a more
or less narrow range. Some form of tuberculosis attacks nearly all
vertebrates, but no other classes of animals and no plants. Lockjaw or
tetanus attacks most mammals, but not any other vertebrates naturally.
Typhoid fever affects human beings; hog cholera, swine, etc. This point
is more fully discussed in Chapter XXIII but can not be too greatly
insisted upon.
“The greatest enemy to mankind is man.”
Exceptions to this statement do occur and are important and must be
considered in efforts to protect completely human beings from disease
(tuberculosis from cattle, glanders from horses, poisoning from spoiled
canned goods, anthrax from hair, hides, wool, of animals dead of the
disease), but the most common human diseases are derived from other
human beings directly or indirectly.
Diseases which are due to unicellular pathogenic microörganisms are
called _infectious_ diseases, while if such diseases are transmitted
under natural conditions from organism to organism they are spoken of
as _contagious_ diseases. Most infectious diseases are contagious but
not all. Tetanus is a good illustration of a non-contagious infectious
disease. There are very few such diseases.
When a unicellular microörganism gains entrance into the body and
brings about any pathological changes there, the result is an
_infection_. Undoubtedly many pathogenic organisms get into the body
but never manifest their presence by causing disease conditions, hence
do not cause an infection. It is the pathological conditions which
result that constitute the infection, and not the mere _invasion_.
The time that elapses between the entrance of the organism and the
appearance of symptoms is called the _period of incubation_ and varies
greatly in different diseases.
The term _infestation_ is used to denote pathological conditions due to
_multicellular_ parasites. Thus an animal is _infested_ (not infected)
with tapeworms, roundworms, lice, mites, etc. Many of these conditions,
probably all, are contagious, _i.e._, transmissable naturally from
animal to animal. The word _contagious_ has been used in a variety
of ways to mean _communicated by direct contact_, communicated by a
living something (_contagium_) that might be carried to a distance
and finally _communicable_ in any manner, transmissable. The agency
of transmission may be very roundabout--as through a _special tick_
in Texas fever, a _mosquito_ in malaria, etc.,--or by direct personal
contact, as generally in venereal diseases. After all, though exactness
is necessary, it is better to learn all possible about the _means of
transmission of diseases_, than quibble as to the terms to be used.
An infectious disease may be _acute_ or _chronic_. An acute infection
is one which runs for a relatively short time and is “self-limited,”
so-called, _i.e._, the organisms cease to manifest their presence after
a time. In some acute infections the time is very short--German measles
usually runs five or six days. Typhoid fever may continue eight to
ten weeks, sometimes longer, yet it is an acute infectious disease.
It is not so much the time as the fact of _self-limitation_ that
characterizes acute infections.
In chronic infections there is little or no evidence of limitation of
the progress of the disease which may continue for years. Tuberculosis
is usually chronic. Leprosy in man is practically always so. Glanders
in horses is most commonly chronic; in mules and in man it is more apt
to be acute.
Many infections begin acutely and later change to the chronic type.
Syphilis in man is a good illustration.
The differences between acute and chronic infections are partly due
to the nature of the organism, partly to the number of organisms
introduced and the point of their introduction and partly to the
resistance of the animal infected.
An infectious disease is said to be _specific_ when one kind of
organism is responsible for its manifestations--as diphtheria due
to the _Corynebacterium diphtheriæ_, lockjaw due to _Clostridium
tetani_, Texas fever due to the _Piroplasma bigeminum_, etc. It is
_non-specific_ when it may be due to a variety of organisms, as
_enteritis_ (generally), _bronchopneumonia_, _wound infections_.
Henle, as early as 1840, stated certain principles that must be
established before a given organism can be accepted as the cause of a
specific disease. These were afterward restated by Koch, and have come
to be known as “Koch’s postulates.” They may be stated as follows:
1. The given organism must be found in all cases of the disease in
question.
2. No other organism must be found in all cases.
3. The organism must, when obtained in pure culture, reproduce the
disease in susceptible animals.
4. It must be recovered from such animals in pure culture and this
culture likewise reproduce the disease.
These postulates have not been fully met with reference to any disease,
but the principles embodied have been applied as far as possible
in all those infections which we recognize as specific, and whose
causative agent is accepted. In many diseases recognized as infectious
and contagious no organism has been found which is regarded as the
specific cause. In some of these the organism appears to be too small
to be seen with the highest powers of the microscope, hence they are
called “_ultramicroscopic_” organisms. Because these agents pass
through the finest bacterial filters, they are also frequently called
“_filterable_.” The term “_virus_” or “_filterable virus_” is likewise
applied to these “ultramicroscopic” and “filterable” agents.
The term _primary infection_ is sometimes applied to the first
manifestation of a disease, either specific or non-specific, while
_secondary_ refers to later developments. For example, a _secondary_
general infection may follow a _primary_ wound infection, or _primary_
lung tuberculosis be followed by _secondary_ generalized tuberculosis,
or _primary_ typhoid fever by a _secondary_ typhoid pneumonia. The
terms _primary_ and _secondary_ are also used where the body is
invaded by one kind of an organism and later on by another kind; thus
a _primary_ measles may be followed by _secondary_ infection of the
middle ear, or a _primary_ influenza may be followed by a _secondary_
pneumonia, or a _primary_ scarlet fever by a _secondary_ nephritis
(inflammation of the kidney). Where several organisms seem to be
associated simultaneously in causing the condition then the term _mixed
infection_ is used--in severe diphtheria, streptococci are commonly
associated with the _Corynebacterium diphtheriæ_. In many cases of
hog-cholera, mixed infections in the lungs and in the intestines are
common. Wound infections are usually _mixed_. _Auto-infection_ refers
to those conditions in which an organism commonly present in or on the
body in a latent or harmless condition gives rise to an infectious
process. If the _Bacterium coli_ normal to the intestine escapes into
the peritoneal cavity, or passes into the bladder, a severe peritonitis
or cystitis, respectively, is apt to result. “Boils” and “pimples”
are frequently autoinfections. Such infections are also spoken of as
_endogenous_ to distinguish them from those due to the entrance of
organisms from without--_exogenous_ infections. _Relapses_ are usually
instances of autoinfection.
Those types of _secondary infection_ where the infecting agent is
transferred from one disease focus to another or several other points
and sets up the infection there are sometimes called _metastases_. Such
are the transfer of tubercle bacilli from lung to intestine, spleen,
etc., the formation of abscesses in internal organs following a primary
surface abscess, the appearance of glanders nodules throughout various
organs following pulmonary glanders, etc.
The characteristic of a pathogenic microörganism which indicates
its ability to cause disease is called its _virulence_. If slightly
virulent, the effect is slight; if highly virulent, the effect is
severe, and may be fatal.
On the other hand, the characteristic of the host which indicates
its capacity for infection is called _susceptibility_. If slightly
susceptible, infection is slight, if highly susceptible, the infection
is severe.
Evidently the degree of infection is dependent in large measure on
the relation between the _virulence_ of the invading organism and the
_susceptibility_ of the host. High virulence and great susceptibility
mean a severe infection; low virulence and little susceptibility a
slight infection; while high virulence and little susceptibility or
low virulence and great susceptibility might mean a moderate infection
varying in either direction. Other factors influencing the degree of
infection are the number of organisms introduced, the point where they
are introduced and various conditions. These will be discussed in
another connection (Chapter XXV).
The study of pathogenic bacteriology includes the thorough study
of the individual organisms according to the methods already given
(Chapters XVIII-XXI) as an aid to diagnosis and subsequent treatment,
bacteriological or other, in a given disease. Of far greater
importance than the _treatment_, which in most infectious diseases
is not specific, is the _prevention_ and _ultimate eradication_ of
all infectious diseases. To accomplish these objects involves further
a study of the _conditions under which pathogenic organisms exist
outside the body_, _the paths of entrance into and elimination from
the body_ and those _agencies within the body itself_ which make it
_less susceptible to infection or overcome the infective agent after
its introduction_. That condition of the body itself which prevents any
manifestation of a virulent pathogenic organism after it has been once
introduced is spoken of as _immunity_ in the modern sense. Immunity is
thus the opposite of susceptibility and may exist in varying degrees.
That scientists are and have been for some years in possession of
sufficient knowledge to permit of the prevention and eradication
of most, if not all, of our infectious diseases can scarcely be
questioned. The practical application of this knowledge presents
many difficulties, the chief of which is the absence of a public
sufficiently enlightened to permit the expenditure of the necessary
funds. Time and educative effort alone can surmount this difficulty. It
will probably be years yet, but it will certainly be accomplished.
CHAPTER XXIII.
PATHOGENIC BACTERIA OUTSIDE THE BODY.
Pathogenic bacteria may exist outside the body of the host under a
variety of conditions as follows:
I. In or on inanimate objects or material.
(_a_) As true saprophytes.
(_b_) As facultative saprophytes.
(_c_) Though obligate parasites, they exist in a latent
state.
II. In or on other animals, or products from them:
A. Susceptible to the disease.
(_a_) Sick themselves.
(As far as human beings are concerned these are
mainly:
1. Other human beings for most diseases.
2. Rats for plague.
3. Dogs for rabies.
4. Horses for glanders.
5. Cattle, swine, parrots for tuberculosis).
(_b_) Recovered from illness.
(_c_) Never sick but “carriers.”
B. Not susceptible.
(_d_) Accidental carriers.
(_e_) Serving as necessary intermediate hosts for certain
stages of the parasite--this applies to _protozoal_
diseases only, as yet.
I.
(_a_) The bacilli of tetanus, malignant edema and the organisms of
“gas gangrene” are widely distributed. There is no evidence that their
entrance into the body is at all necessary for the continuation of
their life processes, or that one case of either of these diseases
ever has any connection with any other case; they are true saprophytes.
Manifestly it would be futile to attempt to prevent or eradicate such
diseases by attacking the organism in its natural habitat. _Clostridium
botulinum_, which causes a type of food poisoning in man, does not even
multiply in the body, but the disease symptoms are due to a soluble
toxin which is produced during its growth outside the body.
(_b_) Organisms like the bacterium of anthrax and the bacillus of
black-leg from their local occurrence seem to be distributed from
animals infected, though capable of a saprophytic existence outside the
body for years. These can no more be attacked during their saprophytic
existence than those just mentioned. Doubtless in warm seasons of the
year and in the tropics other organisms pathogenic to animals may live
and multiply in water or in damp soil where conditions are favorable,
just as the cholera organism in India, and occasionally the typhoid
bacillus in temperate climates do.
(_c_) Most pathogenic organisms, however, when they are thrown off
from the bodies of animals, remain quiescent, do not multiply, in fact
always tend to die out from lack of all that is implied in a “favorable
environment,” food, moisture, temperature, light, etc. Disinfection is
sometimes effective in this class of diseases in preventing new cases.
II. A.
(_a_) The most common infectious diseases of animals are transmitted
more or less directly from other animals of the same species. Human
beings get nearly all their diseases from other human beings who are
sick; horses, from other horses; cattle, from other cattle; swine,
from swine, etc. Occasionally transmission from one species to another
occurs. Tuberculosis of swine most frequently results from feeding
them milk of tuberculous cattle or from their eating the droppings of
such cattle. Human beings occasionally contract anthrax from wool,
hair and hides of animals dead of the disease or from postmortems
on such animals; glanders from horses; tuberculosis (in children)
from tuberculous milk; bubonic plague from rats; rabies practically
always from the bites of dogs and other rabid animals, etc. The
mode of limiting this class of diseases is evidently to isolate the
sick, disinfect their discharges and their _immediate_ surroundings,
sterilize such products as must be handled or used, kill lower animals
that are dangerous, and disinfect, bury properly, or destroy their
carcasses.
Classes of the sick that are especially dangerous for the spread of
disease are the mild cases and the undetected cases. These individuals
do not come under observation and hence not under control.
(_b_) This class of carriers offers a difficult problem in the
prevention of infectious diseases since they may continue to give off
the organisms indefinitely and thus infect others. Typhoid carriers
have been known to do so for fifty-five years. Cholera, diphtheria,
meningitis and other carriers are well known in human practice.
Carriers among animals have not been so frequently demonstrated,
but there is every reason for thinking that hog-cholera, distemper,
roup, influenza and other carriers are common. Carriers furnish the
explanation for many of the so-called “spontaneous” outbreaks of
disease among men and animals.
It is the general rule that those who are sick cease to carry the
organisms on recovery and it is the occasional ones who do not that are
the exceptions. In those diseases in which the organism is known it
can be determined by examination of the patient or his discharges how
long he continues to give off the causative agent. In those in which
the cause is unknown (in human beings, the commonest and most easily
transmitted diseases, scarlet fever, measles, German measles, mumps,
chicken-pox, small-pox, influenza), no such check is possible. It is
not known how long such individuals remain carriers. Hence isolation
and quarantine of such convalescents is based partly on experience
and partly on theory. It is highly probable that in the diseases just
mentioned transmission occurs in the _early stages only_, except
in small-pox and chicken-pox where the organism seems to be in the
pustules and transmission by means of material from these is possible,
though only by direct contact with it.
The fact that such individuals are _known to have had the disease_ is a
guide for control. The methods to be used are essentially the same as
for the sick, (_a_), though obviously such human carriers are much more
difficult to deal with since they are well.
(_c_) Another class of carriers is those who have never had the
disease. Such individuals are common and are very dangerous sources
of infection. Many of them have _associated with the sick or with
convalescents_ and these should always be suspected of harboring the
organisms. Their control differs in no way from that of class (_b_).
Unfortunately a history of such association is too often not available.
Modern transportation and modern social habits are largely responsible
for the nearly universal distribution of this type of carrier. Their
detection is probably the largest single problem in the prevention
of infectious diseases. A partial solution would be universal
bacteriological examination. In our present stage of progress this is
impossible and would not detect carriers of diseases of unknown cause.
The various classes of carriers just discussed are in a large part
responsible for the continued presence of the commoner diseases
throughout the country. The difficulties in control have been
mentioned. A complete solution of the problem is not yet obtained. The
army experience of the past few years in the control of infectious
diseases shows what may be done.
There is another class of carriers which might be called the “universal
carrier,” _i.e._, there are certain organisms which seem to be
constantly or almost constantly present in or on the human body. These
are _micrococci_, _streptococci_ and _pneumococci_, all _Gram positive_
organisms. They are ordinarily harmless parasites, but on occasion may
give rise to serious, even fatal, infection. Infected wounds, pimples,
boils, “common colds,” most “sore throats,” bronchitis, pneumonia are
pathological conditions that come in this class. Such infections are
usually autogenous. There is a constant interchange of these organisms
among individuals closely associated, so that all of a group usually
harbor the same type though no one individual can be called _the_
carrier. Whenever, for any reason, the resistance of an individual (see
Chaps. XXV et seq.) is lowered either locally or generally some of
these organisms are liable to gain a foothold and cause infection. It
sometimes happens that a strain of dangerous organisms may be developed
in an individual in this way which is passed around to others with its
virulence increased and thus cause an epidemic. Or, since all of the
group are living under the same conditions the resistance of all or
many of them may be lowered from the same general cause and an epidemic
result from the organism common to all (pneumonia after measles,
scarlet fever and influenza in camps). Protection of the individual is
chiefly a personal question, _i.e._, by keeping up the “normal healthy
tone” in all possible ways: The use of protective vaccines (Chap. XXX)
appears to be advisable in such instances (colds, pneumonia after
measles and influenza, inflammation of throat and middle ear following
scarlet fever and measles). Results obtained in this country during
the recent influenza epidemic have been conflicting but on the whole
appear to show that preventive vaccination against _pneumonia liable to
follow_ should be practiced.
It would seem that among groups of individuals where infection may be
expected the proper procedure would be to prepare autogenous vaccines
(Chapter XXX) from members of the group and vaccinate all with the
object of protecting them.
II. B.
(_d_) In this class come the “accidental carriers” like flies, fleas,
lice, bed-bugs, ticks, and other biting and blood-sucking insects,
vultures, buzzards, foxes, rats, and carrion-eating animals generally;
pet animals in the household, etc. Here the animals are not susceptible
to the given disease but become contaminated with the organisms
and then through defilement of the food or drink or contact with
individuals or with utensils pass the organisms on to the susceptible.
Some biting and blood-sucking insects transmit the organisms through
biting infected and non-infected animals successively. The spirilloses
and trypanosomiases seem to be transmitted in this way, though there
is evidence accumulating which may place these diseases in the next
class. Anthrax is considered in some instances to be transmitted by
flies and by vultures in the southern United States. Transmission
of typhoid, dysentery, cholera and other diseases by flies is well
established in man. Why not hog-cholera from farm to farm by flies,
English sparrows, pigeons feeding, or by turkey buzzards? Though this
would not be easy to prove, it seems reasonable.
Preventing contact of such animals with the discharges or with the
carcasses of those dead of the disease, destruction of insect carriers,
screening and prevention of fly breeding are obvious protective
measures.
(_e_) In this class come certain diseases for which particular
insects are necessary for the parasite in question, so that certain
stages in its life history may be passed therein. The surest means
for eradicating such diseases is the destruction of the insects
concerned. Up to the present no _bacterial_ disease is known in which
this condition exists, unless Rocky Mountain spotted fever and typhus
fever shall prove to be due to bacteria. Such diseases are all due to
protozoa. Among them are Texas fever, due to _Piroplasma bigeminum_
in this country which has been eradicated in entire districts by
destruction of the cattle tick (_Margaropus annulatus_).
Piroplasmoses in South Africa among cattle and horses, and in other
countries are transmitted in similar ways. Probably many of the
diseases due to spirochetes and trypanosomes are likewise transmitted
by _necessary_ insect intermediaries. In human medicine the eradication
of yellow fever from Panama and Cuba is due to successful warfare
against, a certain mosquito (_Stegomyia_). So the freeing of large
areas in different parts of the world from _malaria_ follows the
destruction of the mosquitoes. The prevention of typhus fever and
of trench fever by “delousing” methods is familiar from recent army
experience though for typhus this method has been practiced in Russia
for more than ten years to the author’s personal knowledge. The
campaign against disease in animals and man from insect sources must be
considered as still in its infancy. The full utilization of tropical
lands depends largely on the solution of this problem.
CHAPTER XXIV.
PATHS OF ENTRANCE OF PATHOGENIC ORGANISMS,
OR
CHANNELS OF INFECTION.
_A._ =The Skin.=--If the skin is healthy there is no opportunity for
bacteria to penetrate it. It is protected not only by the stratified
epithelium, but also in various animals, by coats of hair, wool,
feathers, etc. The secretion pressure of the healthy sweat and oil
glands acts as an effective bar even to motile bacteria. Nevertheless a
very slight injury only is sufficient to give normal surface parasites
and other pathogenics, accidentally or purposely brought in contact
with it, an opportunity for more rapid growth and even entrance
for general infection. Certain diseases due to higher fungi are
characteristically “skin diseases” and rarely become general--various
forms of favus, trichophyton infections, etc. A few disease organisms,
tetanus, malignant edema, usually get in through the skin; others,
black-leg, anthrax, quite commonly; and those diseases transmitted
by biting and blood-sucking insects, piroplasmoses, trypanosomiases,
spirilloses, scarcely in any other way. Defective secretion in the skin
glands from other causes, may permit lodgment and growth of bacteria
in them or in the hair follicles. “Pimples” and boils in man and local
abscesses occasionally in animals are illustrations. Sharp-edged and
freely bleeding wounds are less liable to be infected than contusions,
ragged wounds, burns, etc. The flowing blood washes out the wound and
the clotting seals it, while there is less material to be repaired
by the leukocytes and they are free to care for invading organisms
(phagocytosis). Pathogenic organisms, especially pus cocci, frequently
gain lodgment in the _milk glands_ and cause local (mastitis) or
general infection.
_B._ =Mucosæ directly continuous with the skin and lined with
stratified epithelium= are commonly well protected thereby and by the
secretions.
(_a_) The external auditory meatus is rarely the seat even of local
infection. The tympanic cavity is normally sterile, though it may
become infected by extension through the Eustachian tube from the
pharynx (_otitis media_).
(_b_) The conjunctiva is frequently the seat of localized, very rarely
the point of entrance for a generalized infection, except after severe
injury. Those diseases whose path of entrance is generally assumed to
be the respiratory tract (see “Lungs” below) might also be admitted
through the eye. Material containing such organisms might get on the
conjunctiva and be washed down through the lachrymal canal into the
nose. Experiment has shown that bacteria may pass in this way in a
few minutes. In case masks are worn to avoid infection from patients
suffering with these diseases, the eyes should therefore be protected
as well as the nose and mouth.
(_c_) The nasal cavity on account of its anatomical structure retains
pathogenic organisms which give rise to local infections more
frequently than other mucosæ of its character. These may extend from
here to middle ear, neighboring sinuses, or along the lymph spaces of
the olfactory nerve into the cranial cavity (meningitis). Acute coryza
(“colds” in man) is characteristic. Glanders, occasionally, is primary
in the nose, as is probably roup in chickens, leprosy in man. The
meningococcus and the virus of poliomyelitis pass from the nose into
the cranial cavity without local lesions in the former.
(_d_) The mouth cavity is ordinarily protected by its epithelium
and secretions, though the injured mucosa is a common source of
_actinomycosis_ infection, as well as thrush. In foot-and-mouth disease
no visible lesions seem necessary to permit the localization of the
unknown infective agent.
(_e_) The tonsils afford a ready point of entrance for ever-present
_micrococci_ and _streptococci_ whenever occasion offers (follicular
tonsillitis, “quinsy”), and articular rheumatism is not an uncommon
sequel. The diphtheria bacillus characteristically seeks these
structures for its development. Tubercle and anthrax organisms
occasionally enter here.
(_f_) The pharynx is the seat of localized infection as in
_micrococcal_, _streptococcal_ and diphtherial “sore throat” in human
beings, but both it and the esophagus are rarely infected in animals
except as the result of injury.
(_g_) The external genitalia are the usual points of entrance for
the venereal organisms in man (gonococcus, _Treponema pallidum_, and
Ducrey’s bacillus). The bacillus of contagious abortion and probably
the trypanosome of dourine are commonly introduced through these
channels in animals.
_C._ =Lungs.=--The varied types of pneumonia due to many different
organisms (tubercle, glanders, influenza, plague bacilli, pneumococcus,
streptococcus, micrococcus and many others) show how frequently these
organs are the seat of a localized infection, which may or may not be
general. Whether the lungs are the actual point of entrance in these
cases is a question which is much discussed at the present time,
particularly with reference to tuberculosis. The mucous secretion
of the respiratory tract tends to catch incoming bacteria and other
small particles and the ciliary movement along bronchial tubes and
trachea tends to carry such material out. “Foreign body pneumonia”
shows clinically, and many observers have shown experimentally that
microörganisms may reach the alveoli even though the exchange of
air between them and the bronchioles and larger bronchi takes place
ordinarily only by diffusion. The presence of carbon particles in the
walls of the alveoli in older animals and human beings and in those
that breathe dusty air for long periods indicates strongly, though it
does not prove absolutely, that these came in with inspired air. On the
other hand, experiment has shown that tubercle bacilli introduced into
the intestine may appear in the lungs and cause disease there and not
in the intestine. It is probably safe to assume that in those diseases
which are transmitted most readily through close association though
not necessarily actual contact, the commonest path is through the
respiratory tract, which may or may not show lesions (smallpox, scarlet
fever, measles, chicken-pox, whooping-cough, pneumonic plague in man,
lobar and bronchopneumonias and influenza in man and animals, some
cases of glanders and tuberculosis). On the other hand, the fact that
the _Bacterium typhosum_ and _Bacterium coli_ may cause pneumonia when
they evidently have reached the lung from the intestinal tract, and the
experimental evidence of lung tuberculosis above mentioned show that
this route cannot be excluded in inflammations of the lung.
_D._ =Alimentary Tract.=--The alimentary tract affords the ordinary
path of entrance for the causal microbes of many of the diseases of
animals and man, since they are carried into the body most commonly and
most abundantly in the food and drink.
(_a_) The stomach is rarely the seat of local infection, even in
ruminants, except as the result of trauma. The character of the
epithelium in the rumen, reticulum and omasum in ruminants, the
hydrochloric acid in the abomasum and in the stomachs of animals
generally are usually sufficient protection. Occasionally anthrax
“pustules” develop in the gastric mucosa. (The author saw nine such
pustules in a case of anthrax in a man.)
(_b_) The intestines are frequently the seat of localized infections,
as various “choleras” and “dysenteries” in men and many animals,
anthrax, tuberculosis, Johne’s disease. Here doubtless enter the
organisms causing “hemorrhagic septicemias” in many classes of animals,
and numerous others. These various organisms must have passed through
the stomach and the question at once arises, why did the HCl not
destroy them? It must be remembered that the acid is present only
during stomach digestion, and that liquids taken on an “empty stomach”
pass through rapidly and any organisms present are not subjected to
the action of the acid. Also spores generally resist the acid. Other
organisms may pass through the stomach within masses of undigested
food. The fact that digestion is going on in the stomach of ruminants
practically all the time may explain the relative freedom of _adult_
animals of this class from “choleras” and “dysenteries.”
MECHANISM OF ENTRANCE OF ORGANISMS.
In the preceding chapters statements have been made that “bacteria
enter” at various places or they “pass through” different mucous
membranes, skin, etc. Strictly speaking such statements are
incorrect--bacteria do not “enter” or “pass through” of themselves.
It is true that some of the intestinal organisms are motile, but most
of the bacteria which are pathogenic are non-motile. Even the motile
ones can not make their way against fluids secreted or excreted on free
surfaces. Bacteria cannot pass by diffusion through membranes since
they are finite particles and not in solution.
In the case of penetrating wounds bacteria may be carried mechanically
into the tissues, but this is exceptional in most infections. Also
after gaining lodgment they may gradually grow through by destroying
tissue as they grow, but this is a minor factor. Evidently, there
must be some mechanism by which they _are carried_ through. The known
mechanisms for this in the body are ameboid cells, especially the
phagocytes. It is most probable that these are the chief agents in
getting bacteria into the tissues through various free surfaces. The
phagocytes engulf bacteria, carry them into the tissues and either
destroy them, are destroyed by them, or may disgorge or excrete them
free in the tissues or in the blood.
DISSEMINATION OF ORGANISMS.
Dissemination of organisms within the tissues occurs either through
the lymph channels or the bloodvessels or both. If through the lymph
vessels only it is usually much more restricted in extent, or much more
slowly disseminated, while blood dissemination is characterized by the
number of organs involved simultaneously.
PATHS OF ELIMINATION OF PATHOGENIC MICRÖORGANISMS.
I. Directly from the point, of injury. This is true in infected
wounds open to the surface, skin glanders (farcy), black-leg,
surface anthrax, exanthemata in man and animals (scarlet fever (?),
measles (?), smallpox; hog erysipelas, foot-and-mouth disease):
also in case of disease of mucous membranes continuous with the
skin--from nasal discharges (glanders), saliva (foot-and-mouth
disease), material coughed or sneezed out (tuberculosis, influenza,
pneumonias), urethral and vaginal discharges (gonorrhea and syphilis
in man, contagious abortion and dourine in animals), intestinal
discharges (typhoid fever, “choleras,” “dysenteries,” anthrax,
tuberculosis, Johne’s disease). Material from nose, mouth and lungs
may be swallowed and the organisms passed out through the intestines.
II. Indirectly through the secretions and the excretions where the
internal organs are involved. The _saliva_ of rabid animals contains
the ultramicroscopic virus of rabies (the sympathetic ganglia
within the salivary glands, and pancreas also, are affected in this
disease as well as the cells of the central nervous system). The
_gall-bladder_ in man is known to harbor colon and typhoid bacilli,
as that of hog-cholera hogs does the virus of this disease. It may
harbor analogous organisms in other animals, though such knowledge
is scanty. The _kidneys_ have been shown experimentally to excrete
certain organisms introduced into the circulation within a few minutes
(micrococci, colon and typhoid bacilli, anthrax). Typhoid bacilli occur
in the urine of typhoid-fever patients in about 25 per cent. of all
cases and the urine of hogs with hog cholera is highly virulent. Most
observers are of the opinion, however, that under natural conditions
the kidneys do not excrete bacteria unless they themselves are infected.
The _milk_ both of tuberculous cattle and tuberculous women has been
shown to contain tubercle bacilli _even when the mammary glands are not
involved_. Doubtless such bacteria are carried through the walls of the
secreting tubules or of the smaller ducts by phagocytes and are then
set free in the milk.
SPECIFICITY OF LOCATION OF INFECTIVE ORGANISMS.
It is readily apparent that certain disease organisms tend to locate
themselves in definite regions and the question arises, Is this due
to any specific relationship between organism and tissue or not?
Diphtheria in man usually attacks the tonsils first, gonorrhea and
syphilis the external genitals, tuberculosis the lung, “choleras” the
small intestine, “dysenteries” the large intestine, influenza the
lungs. In these cases the explanation is probably that the points
attacked are the places where the organism is most commonly carried,
with no specific relationship, since all of these organisms (Asiatic
cholera excepted) also produce lesions in other parts of the body _when
they reach them_. On the other hand, the virus of hydrophobia attacks
nerve cells, leprosy frequently singles out nerves, glanders bacilli
introduced into the abdominal cavity of a young male guinea-pig cause
an inflammation of the testicle, malarial parasites and piroplasms
attack the red blood corpuscles, etc. In fact, most _pathogenic
protozoa_ are specific in their localization either in certain tissue
cells or in the blood or lymph. In these cases there is apparently a
real chemical relationship, as there is also between the _toxins_ of
bacteria and certain tissue cells (tetanus toxin and nerve cells).
Whether “chemotherapy” will ever profit from a knowledge of such
chemical relationships remains to be developed. It appears that a
search for these specific chemical substances with the object of
combining poisons with them so that the organisms might in this way be
destroyed, would be a profitable line of research.
CHAPTER XXV.
IMMUNITY.
Immunity, as has already been stated, implies such a condition of the
body that pathogenic organisms after they have been introduced are
incapable of manifesting themselves, and are unable to cause disease.
The word has come to have a more specific meaning than resistance in
many instances, in other cases the terms are used synonymously. It is
the opposite of susceptibility. The term must be understood always in a
relative sense, since no animal is immune to all pathogenic organisms,
and conceivably not entirely so to anyone, because there is no question
that a sufficient number of bacteria of any kind might be injected
into the circulation to kill an animal, even though it did it purely
mechanically.
Immunity may be considered with reference to a single individual or to
entire divisions of the organic world, with all grades between. Thus
plants are immune to the diseases affecting animals; invertebrates to
vertebrate diseases; cold-blooded animals to those of warm blood; man
is immune to most of the diseases affecting other mammals; the rat to
anthrax, which affects other rodents and most mammals; the well-known
race of Algerian sheep is likewise immune to anthrax while other sheep
are susceptible; the negro appears more resistant to yellow fever than
the white; some few individuals in a herd of hogs always escape an
epizoötic of hog cholera, etc.
Immunity within a given species is modified by a number of
factors--age, state of nutrition, extremes of heat or cold, fatigue,
excesses of any kind, in fact, anything which tends to lower the
“normal healthy tone” of an animal also tends to lower its resistance.
Children appear more susceptible to scarlet fever, measles,
whooping-cough, etc., than adults; young cattle more frequently have
black-leg than older ones (these apparently greater susceptibilities
may be due in part to the fact that most of the older individuals
have had the diseases when young and are immune for this reason).
Animals weakened by hunger or thirst succumb to infection more readily.
Frogs and chickens are immune to tetanus, but if the former be put
in water and warmed up to and kept, at about 37°, and the latter be
chilled for several hours in ice-water, then each may be infected.
Pneumonia frequently follows exposure to cold. The immune rat may
be given anthrax if first he is made to run in a “squirrel cage”
until exhausted. Alcoholics are far less resistant to infection than
temperate individuals. “Worry,” mental anguish, tend to predispose to
infection.
The following outlines summarize the different, classifications of
immunity so far as mammals are concerned for the purposes of discussion.
Immunity.
{ {1. Inherited through
{ { the germ cell or cells.
{ { {(_a_) By having the
{A. Congenital {2. Acquired { disease _in utero_.
I. Natural { { _in utero_. {(_b_) By absorption
{ { { of immune
{ { substances
{B. Acquired by { from the mother.
{ having the disease.
II. Artificial--acquired through human agency by:
1. Introduction of the organism or its products.
2. Introduction of the blood serum of an immune animal.
Immunity.
I. Active--due to the introduction of the organism or due to the
introduction of the products of the organism.
A. Naturally by having the disease.
B. Artificially.
1. By introducing the organism:
{1. Passage through another animal.
{2. Drying.
(_a_) Alive and virulent. {3. Growing at a higher temperature.
(_b_) Alive and virulence {4. Heating the cultures.
reduced by {5. Treating with chemicals.
(_c_) Dead. {6. Sensitizing.
{7. Cultivation on artificial media.
2. By introducing the products of the organism.
II. Passive--due to the introduction of the blood serum of an actively
immunized animal.
Immunity present in an animal and not due to human interference is to
be regarded as _natural_ immunity, while if brought about by man’s
effort it is considered _artificial_. Those cases of natural immunity
mentioned above which are common to divisions, classes, orders,
families, species or races of organisms and to those few individuals
where no special cause is discoverable, must be regarded as instances
of true _inheritance_ through the germ cell as other characteristics
are. All other kinds of immunity are _acquired_. Occasionally young are
born with every evidence that they have had a disease _in utero_ and
are thereafter as immune as though the attack had occurred after birth
(“small-pox babies,” “hog-cholera pigs”). Experiment has shown that
immune substances may pass from the blood of the mother to the fetus
_in utero_ and the young be immune for a time after birth (tetanus).
This is of no practical value as yet. It is a familiar fact that with
most infectious diseases recovery from one attack confers a more or
less lasting immunity, though there are marked exceptions.
=Active Immunity.=--By active immunity is meant that which is due
to the actual introduction of the organism, or in some cases of its
products. The term active is used because the body cells of the animal
immunized perform the real work of bringing about the immunity as
will be discussed later. In _passive_ immunity the blood serum of an
actively immunized animal is introduced into a second animal, which
thereupon becomes immune, though its cells are not concerned in the
process. The animal is _passive_, just as a test-tube, in which a
reaction takes place, plays no other part than that of a passive
container for the reagents.
In _active_ immunity the organism may be introduced in what is to
be considered a natural manner, as when an animal becomes infected,
has a disease, without human interference. Or the organism may be
purposely introduced to bring about the immunity. For certain purposes
the introduction of the products of the organism (toxins) is used to
bring about active immunity (preparation of diphtheria and tetanus
antitoxin from the horse). The method of producing active immunity by
the artificial introduction of the organism is called _vaccination_,
and a _vaccine_ must therefore contain the organism. _Vaccines_ for
_bacterial_ diseases are frequently called _bacterins_. The use of
the blood serum of an immunized animal to confer passive immunity on
a second animal is properly called _serum therapy_, and the serum so
used is spoken of as an _antiserum_, though the latter word is also
used to denote any serum containing any kind of an antibody (Chapters
XXVII-XXXI). In a few instances both the organism and an antiserum are
used to cause both active and passive immunity (_serum-simultaneous
method_ in immunizing against hog cholera).
In producing active immunity the organism may be introduced (_a_)
_alive and virulent_, but in very small doses, or in combination with
an immune serum, as just mentioned for hog cholera. The introduction
of the live virulent organism alone is done only experimentally as
yet, as it is obviously too dangerous to do in practice, except under
the strictest control (introduction of a _single tubercle_ bacillus,
followed by gradually increasing numbers--Barber and Webb). More
commonly the organisms are introduced (_b_) alive but with their
_virulence reduced_ (“attenuated”) in one of several ways: (1) By
passing the organism through another animal as is the case with
_smallpox vaccine_ derived from a calf or heifer. This method was
first introduced by Jenner in 1795 and was the first practical means
of preventing disease by _vaccination_. This word was used because
material was derived from a cow--Latin _vacca_. (2) By drying the
organism, as is done in the preparation of the vaccine for the _Pasteur
treatment of rabies_, where the spinal cords of rabbits are dried for
varying lengths of time--one to four days, Russian method, one to three
days, German method, longer in this country. (It is probable that the
passage of the “fixed virus” through the rabbit is as important in this
procedure as the drying, since it is doubtful if the “fixed virus” is
pathogenic for man.) It would be more correct to speak of this as a
_preventive vaccination against rabies_, since the latter is one of the
few diseases which is not amenable to _treatment_. The patient always
dies if the disease develops. (3) The organism may be attenuated by
growing at a temperature above the normal. This is the method used in
preparing _anthrax vaccine_ as done by Pasteur originally. (4) Instead
of growing at a higher temperature the culture may be heated in such
a way that it is not killed but merely weakened. _Black-leg_ vaccines
are made by this method. (5) Chemicals are sometimes added to attenuate
the organisms, as was formerly done in the preparation of black-leg
vaccine by Kruse’s method in Germany. The use of toxin-antitoxin
mixtures in immunizing against diphtheria and in the preparation
of diphtheria antitoxin from horses is an application of the same
principle, though here it is the _product_ of the organism and not the
organism whose action is weakened. (6) Within the past few years the
workers in the Pasteur Institute in Paris have been experimenting with
vaccines prepared by treating living virulent bacteria with antisera
(“sensitizing them”) so that they are no longer capable of causing
the disease when introduced, but do cause the production of an active
immunity. The method has been used with typhoid fever bacilli in man
and seems to be successful. It remains to be tried out further before
its worth is demonstrated (the procedure is more complicated and the
chance for infection apparently much greater than by the use of killed
cultures). The term _sero-bacterins_ is used by manufacturers in this
country to designate such bacterial vaccines. (7) Growing on artificial
culture media reduces the virulence of most organisms after a longer
or shorter time. This method has been tried with many organisms in the
laboratory, but is not now used in practice. The difficulties are that
the attenuation is very uncertain and that the organisms tend to regain
their virulence when introduced into the body.
In producing active immunity against many bacterial diseases the
organisms are introduced (_c_) dead. They are killed by heat or by
chemicals, or by using both methods (Chapter XXX).
When the products of an organism are introduced the resulting immunity
is against the products only and not against the organism. If the
organism itself is introduced there results an immunity against it
and in some cases also against the products, though the latter does
not necessarily follow. Hence the immunity may be _antibacterial_ or
_antitoxic_ or both.
Investigation as to the causes of immunity and the various methods by
which it is produced has not resulted in the discovery of specific
methods of treatment for as many diseases as was hoped for at one
time. Just at present progress in serum therapy appears to be at a
standstill, though vaccines are giving good results in many instances
not believed possible a few years ago. As a consequence workers in all
parts of the world are giving more and more attention to the search for
_specific chemical substances_, which will destroy invading parasites
and not injure the host (_chemotherapy_). Nevertheless, in the study
of immunity very much of value in the treatment and prevention of
disease has been learned. Also much knowledge which is of the greatest
use in other lines has been accumulated. Methods of _diagnosis_ of
great exactness have resulted, applicable in numerous diseases. Ways
of _detecting adulteration_ in foods, particularly foods from animal
sources, and of _differentiating proteins_ of varied origin, as well
as means of establishing _biological relationships_ and differences
among groups of animals through “immunity reactions” of blood serums
have followed from knowledge gained by application of the facts or the
methods of immunity research. Hence the study of “immunity problems”
has come to include much more than merely the study of those factors
which prevent the development of disease in an animal or result in
its spontaneous recovery. A proper understanding of the principles of
immunity necessitates a study of these various features and they will
be considered in the discussion to follow.
CHAPTER XXVI.
THEORIES OF IMMUNITY.
Pasteur and the bacteriologists of his time discovered that bacteria
cease to grow in artificial culture media after a time, because of
the exhaustion of the food material in some cases and because of the
injurious action of their own products in other instances. These facts
were brought forward to explain immunity shortly after bacteria were
shown to be the cause of certain diseases. Theories based on these
observations were called (1) “_Exhaustion Theory_” of _Pasteur_, and
(2) “_Noxious Retention Theory_” of _Chauveau_ respectively. The fact,
soon discovered, that virulent pathogenic bacteria are not uncommonly
present in perfectly healthy animals, and the later discovery that
immunity may be conferred by the injection of dead bacteria have led
to the abandonment of both these older ideas. The (3) “_Unfavorable
Environment_” theory of _Baumgartner_, _i.e._, bacteria do not grow
in the body and produce disease because their surroundings are not
suitable, in a sense covers the whole ground, though it is not true as
to the first part, as was pointed out above, and is of no value as a
working basis, since it offers no explanation as to _what the factors
are_ that constitute the “_unfavorable environment_.” Metchnikoff
brought forward a rational explanation of immunity with his (4)
“_Cellular or Phagocytosis Theory_.” As first propounded it based
immunity on the observed fact that certain white blood corpuscles,
_phagocytes_, engulf and destroy bacteria. Metchnikoff has since
elaborated the original theory to explain facts of later discovery.
Ehrlich soon after published his (5) “_Chemical or Side-chain Theory_”
which seeks to explain immunity on the basis of _chemical substances_
in the body which may in part destroy pathogenic organisms or in
part neutralize their products; or in some instances there may be
an absence of certain chemical substances in the body cells so that
bacteria or their products _cannot unite_ with the cells and hence can
do no damage.
[Illustration: PLATE VI
PAUL EHRLICH]
At the present time it is generally accepted, in this country at
least, that Ehrlich’s theory explains immunity in many diseases as
well as many of the phenomena related to immunity, and in other
diseases the phagocytes, frequently assisted by chemical substances,
are the chief factors. Specific instances are discussed in _Pathogenic
Bacteriologies_ which should be consulted. It is essential that the
student should be familiar with the basic ideas of the chemical
theory, not only from the standpoint of immunity, but also in order to
understand the principles of a number of valuable methods of diagnosis.
The chemical theory rests on three fundamental physiological
principles: (1) the response of cells to stimuli, in this connection
_specific chemical stimuli_, (2) the presence within cells of _specific
chemical groups_ which combine with chemical stimuli and thus enable
them to act on the cell, which groups Ehrlich has named _receptors_,
and (3) the “_over-production_” activity of cells as announced by
Weigert.
1. That cells respond to stimuli is fundamental in physiology. These
stimuli may be of many kinds as mechanical, electrical, light, thermal,
chemical, etc. The body possesses groups of cells specially developed
to _receive_ some of these stimuli--touch cells for mechanical stimuli,
retinal cells for light, temperature nerve endings for thermal,
olfactory and gustatory cells for certain chemical stimuli. _Response_
to chemical stimuli is well illustrated along the digestive tract. That
the chemical stimuli in digestion may be more or less specific is shown
by the observed differences in the enzymes of the pancreatic juice
dependent on the relative amounts of carbohydrates, fats, or proteins
in the food, the specific enzyme in each case being increased in the
juice with the increase of its corresponding foodstuff. The cells of
the body, or certain of them at least, seem to respond in a specific
way when substances are brought into direct contact with them, that is,
without having been subjected to digestion in the alimentary tract,
but injected directly into the blood or lymph stream. Cells may be
affected by stimuli in one of three ways: if the stimulus is too weak,
there is no effect (in reality there is no “stimulus” acting); if the
stimulus is too strong, the cell is injured, or may be destroyed; if
the stimulus is of proper amount then it excites the cell to increased
activity, and in the case of _specific chemical stimuli_ the increased
activity, as mentioned for the pancreas, shows itself in an _increased
production of whatever is called forth by the chemical stimulus_. In
the case of many organic chemicals, the substances produced by the
cells under their direct stimulation are markedly specific for the
particular substance introduced.
2. Since chemical action always implies at least two bodies to react,
Ehrlich assumes that in every cell which is affected by a chemical
stimulus there must therefore be a chemical group to unite with this
stimulus. He further states that there must be as many different
kinds of these groups as there are different kinds of chemicals which
stimulate the cell. Since these groups are present in the body cells to
_take up_ different kinds of chemical substances, Ehrlich calls them
_receptors_. Since these groups must be small as compared with the cell
as a whole, and must be more or less on the surface and unite readily
with chemical substances he further speaks of them as “side-chains”
after the analogy of compounds of the aromatic series especially. The
term _receptors_ is now generally used. As was stated above, the effect
of _specific chemical stimuli_ is to cause the production of _more of
the particular substance_ for which it is specific and in the class
of bodies under discussion, the _particular product is these cell
receptors_ with which the chemical may unite.
3. Weigert first called attention to the practically constant
phenomenon that cells ordinarily respond by doing more of a particular
response than is actually called for by the stimulus, that there is
always an “overproduction” of activity. In the case of chemical stimuli
this means an _increased production of the specific substance_ over and
above the amount actually needed.
The student will better understand this theory if he recalls his
fundamental physiology. Living substance is characterized, among other
things, by irritability which is instability. It is in a constant,
state of unstable equilibrium. Whenever the equilibrium becomes
permanently stable the substance is dead. It is also continually
attempting to restore disturbances in its equilibrium. Whenever a
chemical substance unites with a chemical substance in the cell, a
receptor, the latter is, so far as the cell is concerned, _thrown out
of function_ for that cell. The chemical equilibrium of the latter
is upset. It attempts to restore this and does so by making a _new_
receptor to take the place of the one thrown out of function. If this
process is continued, _i.e._, if the new receptor is similarly “used
up” and others similarly formed are also, then the cell will prepare
a supply of these and even an excess, according to Weigert’s theory.
Whenever a cell accumulates an excess of products the normal result
is that it excretes them from its own substance into the surrounding
lymph, whence they reach the blood stream to be either carried to
the true excretory organs, utilized by other cells or remain for a
longer or shorter time in the blood. Hence the excess of receptors
is _excreted from the cell that forms them_ and they become _free_
in the blood. These free receptors are termed _antibodies_. _They
are receptors_ but instead of being retained in the cell are _free
in solution in the blood_. One function of the free receptor, the
antibody, is _always to unite with the chemical substance which caused
it to be formed_. _It may have additional functions._ The chemical
substance which caused the excess formation of receptors, antibodies,
is termed an _antigen_ for that particular kind of antibody.
To recapitulate, Ehrlich’s theory postulates _specific chemical
stimuli_, which react with _specific chemical substances in the body
cells, named receptors_, and that these _receptors_, according to
Weigert, are _produced in excess_ and hence are excreted from the
cell and become _free receptors_ in the blood and lymph. These _free
receptors_ are the various kinds of _antibodies_, the kind depending
on the nature of the stimulus, antigen, the substance introduced. Any
substance which when introduced into the body causes the formation of
an antibody of any kind whatsoever is called an _antigen_,[23] _i.e._,
anti (body) former.
The foregoing discussion explains Ehrlich’s theory of immunity.
According to this theory the _manner of formation of all antibodies_ is
the same. The _kind of antibody_ and the _manner of its action_ will
differ with the _different kinds of antigens_ used.
The succeeding chapters discuss some of the kinds of antibodies, the
theory of their action and some practical applications. It must be
borne in mind throughout the study of these, as has been stated, that
_every antibody has the property of uniting with its antigen whether it
has any property in addition or not_.
Just what antibodies are chemically has not been determined because
no one has as yet succeeded in isolating them chemically pure. To the
author they appear to be enzymes.
Antigens were considered by Ehrlich to be proteins or to be related to
proteins. Most workers since Ehrlich have held similar views. Dr. Carl
Warden of the University of Michigan has been doing much work in recent
years in which he is attempting to show that the antigens are not
proteins but are fats or fatty acids. Mr. E. E. H. Boyer, in his work
(not yet published) in the author’s laboratory for the degree of Ph.D.,
received in June, 1920, succeeded in producing various antibodies from
_Bacterium coli_ antigens. In these antigens he could detect only fatty
acids or salts of fatty acids. If the work of these men is confirmed,
it will open up a most interesting and extremely important field in
immunity and in preventive medicine. It is not apparent that the nature
of the antigen would affect Ehrlich’s theory of the formation of
antibodies.
The author has no doubt that eventually the formation of antibodies and
the reactions between them and their antigens will be explained on the
basis of physical-chemical laws, but this probably awaits the discovery
of their nature.
CHAPTER XXVII.
RECEPTORS OF THE FIRST ORDER.