if time is of importance or acids are used during the
staining process, it is often advisable to add a trace of
Mayer's albumin to the alcohol before floating out the
section. If this substance is employed, a sojourn of twenty
minutes to half an hour in the "hot" incubator will be found
ample to ensure firm adhesion of the section to the slide.
The albuminous fluid is prepared as follows:
[Illustration: FIG. 75.--Section rack.]
~Mayer's Albumin.~--
Weigh out
Salicylate of soda 1 gramme
and dissolve in
Glycerine 50 c.c.
Add
White of egg 50 c.c.
Mix thoroughly by means of an egg whisk.
Filter into a clean bottle.
As an alternative method paint a thin layer of Schallibaum's
solution on the slide with a camel's hair pencil; lay the
section carefully on this film and heat gently to fix the
section.
_Schallibaum's solution_:
Clove oil 30 c.c.
Collodion 10 c.c.
Keep in a dark blue bottle in a cool place.
~Staining Paraffin Sections.~--
1. Warm paraffin section over the Bunsen flame to soften (_but not to
melt_) the paraffin, then dissolve out the wax with xylol poured on from
a drop bottle.
2. Remove xylol by flushing the section with alcohol.
3. If the tissue was originally "fixed" in a corrosive sublimate
solution, the section must now be treated with Lugol's iodine solution
for two minutes and subsequently immersed in 90 per cent. alcohol to
remove all traces of yellow staining.
4. Wash in water.
5. Stain deeply, if using a single stain, as the subsequent processes
decolourise.
6. Wash in water, decolourise if necessary.
7. Flood with several changes of absolute alcohol to dehydrate the
section.
8. Clear in xylol. (Oil of cloves is not usually employed, as it
decolourises the section.)
9. Mount in xylol balsam.
SPECIAL STAINING METHODS FOR SECTIONS.
~Double-staining Carmine and Gram-Weigert.~--
1. Prepare the section for staining as above, sections 1 to 3.
2. Stain in lithium carmine (Orth's) or picrocarmine for ten to thirty
minutes, in a porcelain staining pot (Fig. 76).
3. Wash in picric acid solution until yellow. At this stage cell nuclei
are red, protoplasm is yellow, and bacteria are colourless.
Picric acid solution is prepared by mixing
Picric acid, saturated aqueous solution 40 c.c.
Hydrochloric acid 1 c.c.
Alcohol (90 per cent.) 160 c.c.
4. Wash in water.
5. Wash in alcohol.
6. Stain in aniline gentian violet.
7. Wash in iodine solution till dark brown or black.
8. Wash in water.
9. Dip in absolute alcohol for a second.
10. Decolourise with aniline oil till no more colour is discharged.
[Illustration: FIG. 76.--Staining pot.]
11. Wash with aniline oil, 2 parts, xylol, 1 part.
12. Clear with xylol.
13. Mount in xylol balsam.
~Alternative Gram-Weigert Method for Sections.~--
1. Fix paraffin section on slide and prepare for staining in the usual
manner.
2. Stain in alum carmine for about fifteen minutes.
3. Wash thoroughly in water.
4. Filter aniline gentian violet solution on to the section on the slide
and allow to stain about twenty-five minutes.
5. Wash thoroughly in water.
6. Treat with Lugol's iodine until section ceases to become any blacker.
7. Wash thoroughly in water.
8. Treat with a mixture of equal parts of aniline oil and xylol until no
more colour comes away.
9. Wash thoroughly with xylol.
10. Decolourise and dehydrate rapidly with absolute alcohol until there
remains only a very faint bluish tint.
11. Clear with xylol.
12. Mount in xylol balsam.
(Then fibrin and hyaline tissue are stained deep blue, whilst bacteria
which "stain Gram" appear of a deep blue-violet colour.)
~Unna-Pappenheim Method.~--
Stain.--
Weigh out and mix
Methylene green 0.15 gramme
Pyronin 0.25 gramme
and dissolve in
Carbolic acid 0.5 per cent. aqueous solution 78 c.c.
Measure out
Alcohol 2.5 c.c. }
Glycerine 20.0 c.c. } and add to the stain.
~Method.~--
1. Place tissue in the above stain for ten minutes.
2. Differentiate and dehydrate with absolute alcohol.
3. Clear in xylol.
4. Mount in xylol balsam.
~To Demonstrate Capsules.~--
1. _MacConkey's Method._--Stain precisely as for cover-slip films
(_vide_ page 100).
2. _Friedlaender's Method._--
Stain.--
Gentian violet, saturated alcoholic solution 50 c.c.
Acetic acid, glacial 10 c.c.
Distilled water 100 c.c.
METHOD.--
1. Prepare the sections for staining, _secundum artem_.
2. Stain sections in the warm (e. g., in the hot
incubator) for twenty-four hours.
3. Wash with water.
4. Decolourise lightly with acetic acid, 1 per cent.
5. Dehydrate rapidly with absolute alcohol.
6. Clear with xylol.
7. Mount in xylol balsam.
~To Demonstrate Acid-fast Bacilli.~--
1. Prepare the sections for staining in the usual way.
2. Stain with haematin solution ten to twenty seconds, to obtain a pure
nuclear stain; then wash in water.
3. Stain with carbolic fuchsin twenty to thirty minutes at 47 deg. C.;
then wash in water.
4. Treat with aniline hydrochlorate, 2 per cent. aqueous solution, for
two to five seconds.
5. Decolourise in 75 per cent. alcohol till section appears free from
stain--fifteen to thirty minutes.
6. Dehydrate with absolute alcohol.
7. Clear very rapidly with xylol.
8. Mount in xylol balsam.
~To Demonstrate Spirochaetes in Tissues.~
~Piridin Method (Levaditi).~--
1. Cut slices of tissue 1 mm. thick.
2. Fix in 10 per cent. formalin solution for twenty-four hours.
3. Wash in water for one hour.
4. Place in 96 per cent. alcohol for twenty-four hours.
5. Measure into a dark green or amber bottle 100 c.c. silver nitrate
solution 1 per cent., and 10 grammes pyridin puriss. Transfer slices of
tissue to this. Stopper and keep at room temperature three hours, then
in thermostat at 50 deg. C. for four to six hours.
6. Wash quickly in 10 per cent. pyridin solution.
7. Reduce silver by transferring slices of tissue to following solution
for forty-eight hours.
Pyrogallic acid 4 grammes
Acetone 10 c.c.
Pyridin puriss 15 grammes
Distilled water 100 c.c.
8. Wash well in water.
Take through alcohols of increasing strength up to absolute, keeping in
each strength for twenty-four hours.
9. Clear, embed, cut very thin sections, mount, remove paraffin, again
clear and mount in xylol balsam.
The spirochaetes if present are black and show up against the pale yellow
color of the background.
Weak carbol fuchsin, neutral red or toluidin blue can also be used to
stain the background if desired, after the removal of the paraffin in
step 9.
~To Demonstrate Protozoa in Sections (Leishman).~--
Reagents required:
Leishman's Polychrome stain.
Acetic acid 1 in 1500 aqueous solution.
Caustic soda 1 in 7000 aqueous solution.
Distilled water.
1. Mount section, remove paraffin and take into distilled water as usual
(_vide_ page 121).
2. Drain off the excess of water.
3. Cover the section with diluted Leishman (1 part stain, 2 parts
distilled water) and allow to act for five to ten minutes (until tissue
appears a deep blue).
4. Decolourise with acetic acid solution until only the nuclei appear
blue (examine the section wet, with low power objective).
5. If the eosin colour is too well marked treat with the caustic soda
solution until the desired tint is obtained (as seen with the 1/6-inch
objective).
6. Wash with distilled water.
7. Rapidly dehydrate with alcohol.
8. Clear with xylol.
9. Mount in xylol balsam.
~VIII. CLASSIFICATION OF FUNGI.~
For practical purposes FUNGI may be divided into:
~1. Hymenomycetes~ (including the mushrooms, etc.).
~2. Hyphomycetes~ (moulds).
~3. Blastomycetes~ (yeasts and torulae).
~4. Schizomycetes~ (bacteria).
NOTE.--Formerly myxomycetes were included in the fungi; they
are now recognized as belonging to the animal kingdom, and
are termed "mycetozoa."
~MORPHOLOGY OF THE HYPHOMYCETES.~
At the commencement of his studies, the attention of the student is
directed to the various non-pathogenic moulds and yeasts, not only that
he may gain the necessary technique whilst handling cultivations of
harmless organisms, but also because these very species are amongst the
commonest of those that may accidentally contaminate his future
preparations.
The hyphomycetes are composed of a mycelium of short jointed rods or
"hyphae" springing from an axis or germinal tube which develops from the
spore. Hyphae are--
(a) Nutritive or submerged.
(b) Reproductive or aerial.
The protoplasm of these cells contains granules, pigment, oil globules,
and sometimes crystals of calcium oxalate.
~Reproduction.~--Apical spore formation--asexual;
zoospores--sexual.
~Mucorinae.~--_Mucor_ (Fig. 77).--Note the branching filaments--"mycelium"
(a), "hyphae" (b).
Note the asexual reproduction.
1. A filament grows upward. At its apex a septum forms, then a globular
swelling appears--"sporagium" (d). This possesses a definite membrane.
2. From the septum grows a club-shaped mass of protoplasm--"columella"
(c).
[Illustration: FIG. 77.--Mucor mucedo.]
[Illustration: FIG. 78.--Aspergillus]
3. The rest of the contained protoplasm breaks up into "swarm spores"
(e).
Finally the membrane ruptures and spores escape.
~Perisporaceae.~--_Aspergillus_ (Fig. 78).--Note the branching
filaments--"mycelium" (a).
[Illustration: FIG. 79.--Penicillium.]
Note the asexual reproduction.
1. A filament (b) grows upward, its termination becomes clubbed; on
the clubbed extremity flask-shaped cells appear--"sterigmata" (c).
2. At free end of each sterigma is formed an oval body--a spore or
"gonidium" (d), which, when ripe, is thrown off from the sterigma. Two
or more gonidia may be supported upon each sterigma.
_Penicillium_ (Fig. 79).--Note the branching filaments--"mycelium" (a)
(frequently containing globules).
Note the asexual reproduction.
1. A filament grows upward--"goniodophore" (b)--and its apex divides
up into several branches--"basidia" (c).
2. At the apex of each basidium a flask-shaped cell, "sterigma" (d),
appears.
3. At the apex of each sterigma appears a row of oval cells--"spores" or
"conidia" (e). These, when ripe, are cast off from the sterigmata.
[Illustration: FIG. 80.--Oidium.]
~Ascomycetae.~--_Oidium_ (Fig. 80).--(This family is perhaps as nearly
related to the blastomycetes as it is to the hyphomycetes.)
Note the branching filaments--"pseudomycelium" (a). Here and there
filaments are broken up at their ends into oval or rod-shaped segments,
"oidia," and behave as spores.
Note the asexual reproduction. From the pseudomycelium arise true hyphae
(b), each of which in turn ends in a chain of spores (c).
~MORPHOLOGY OF THE BLASTOMYCETES.~
The blastomycetes are composed of spherical or oval cells (8 to 9.5 mu in
diameter), which, when rapidly multiplying by budding, may form a
spurious mycelium. A thin cell-wall encloses the granular protoplasm, in
which vacuoles and sometimes a nucleus may be noted. This latter is best
seen when stained with haematoxylin (see page 105).
During their growth and multiplication the blastomycetes split up
solutions containing sugar into alcohol and CO_{2}.
~Saccharomyces~ (Fig. 81).--Note the round or oval cells of granular
protoplasm (a) containing solid particles and vacuoles (c), and
surrounded by a definite envelope.
~Reproduction.~--Budding; ascospores--asexual.
Note the asexual _reproduction_.
1. "Gemmation"--that is, the budding out of daughter cells (b) from
various parts of the gradually enlarging mother cell. These are
eventually cast off and in turn become mother cells and form fresh
groups of buds.
[Illustration: FIG. 81.--Saccharomyces with ascospores.]
[Illustration: FIG. 82.--Torula.]
2. Spore formation--"ascospores" (e). These are formed at definite
temperatures and within well-defined periods; e. g., Saccharomyces
cerevisiae, thirty hours at 25 deg. to 37 deg. C., or ten days at
12 deg. C.
~Torulae~ (Fig. 82).--Torulae, whilst resembling yeasts in almost
every other respect, never form endo-spores. Note the elongated,
sausage-shaped cells (a) the larger oval cells (b) and the globular
cells (c) the former two often interlacing and growing as a film.
Note the absence of ascospore formation.
IX. SCHIZOMYCETES.
~Classification and Morphology.~--Bacteria are often classified, in
general terms, according to their life functions, into--
_Saprogenic_, or putrefactive bacteria;
_Zymogenic_, or fermentative bacteria;
_Pathogenic_, or disease-producing bacteria;
or according to their food requirements into--
_Prototrophic_, requiring no organic food (e. g., nitrifying bacteria);
_Metatrophic_, requiring organic food (e. g., saprophytes
and facultative parasites);
_Paratrophic_, requiring living food (obligate parasites);
or according to their metabolic products into--
_Chromogenic_, or pigment-producing bacteria;
_Photogenic_, or light-producing bacteria;
_Aerogenic_, or gas-producing bacteria;
and so on.
Such broad groupings as these have, however, but little practical value
when applied to the systematic study of the fission fungi.
On the other hand, no really scientific classification of the
schizomycetes has yet been drawn up, and the varying morphological
appearances of the members of the family are still utilised as a basis
for classification, as under--
~1. Cocci.~ (Fig. 83).--Rounded or oval cells, subdivided according to the
arrangement of the individuals after fission, into--
_Diplococci_ and _Streptococci_, where division takes place in one plane
only, and the individuals remain attached (a) in pairs or (b) in
chains.
_Tetrads_, _Merismopedia_, or _Pediococci_, where division takes place
alternately in two planes at right angles to each other, and the
individuals remain attached in flat tablets of four, or its multiples.
[Illustration: FIG. 83.--Types of bacteria--cocci: 1, Diagram of sphere
indicating planes of fission; 2, diplococci; 3, streptococci; 4,
tetrads; 5, sarcinae; 6, staphylococci.]
_Sarcinae_, where division takes place in three planes successively,
and the individuals remain attached in cubical packets of eight and its
multiples.
[Illustration: FIG. 84.--Types of bacteria--bacilli, etc.: 1, Bacilli;
2, diplobacilli; 3 streptobacilli; 4, spirilla; 5, vibrios; 6,
spirochaetae.]
_Micrococci_ or _Staphylococci_, where division takes place in three
planes, but with no definite sequence; consequently the individuals
remain attached in pairs, short chains, plates of four, cubical packets
of eight, and irregular masses containing numerous cocci.
~2. Bacilli~ (Fig. 84, 1 to 3).--Rod-shaped cells. A bacillus, however
short, can usually be distinguished from a coccus in that two sides are
parallel. Some bacilli after fission retain a characteristic arrangement
and may be spoken of as _Diplobacilli_ or _Streptobacilli_.
Leptothrix is a term that in the past has been loosely used to signify a
long thread, but is now restricted to such forms as belong to the
leptothriciae (_vide infra_).
~3. Spirilla~ (Fig. 84, 4 to 6).--Curved and twisted filaments.
Classified, according to shape, into--
Spirillum.
Vibrio (comma).
Spirochaeta.
Many Spirochaetes appear to belong to the animal kingdom and are grouped
under protozoa; other organisms to which this name has been given are
undoubtedly bacteria.
Higher forms of bacteria are also met with, which possess the following
characteristics: They are attached, unbranched, filamentous forms,
showing--
(a) Differentiation between base and apex;
(b) Growth apparently apical;
(c) Exaggerated pleomorphism;
(d) "Pseudo-branching" from apposition of cells; and are classified
into--
1. Beggiotoa. } Free swimming forms, which
2. Thiothrix. } contain sulphur granules.
3. Crenothrix. }
4. Cladothrix. } These forms do not contain
5. Leptothrix. } sulphur granules.
6. Streptothrix. A group which exhibits true but
not dichotomous branching, and contains some pathogenic
species.
The morphology of the same bacterium may vary greatly under different
conditions.
For example, under one set of conditions the examination of a pure
cultivation of a bacillus may show a short oval rod as the predominant
form, whilst another culture of the same bacillus, but grown under
different conditions, may consist almost entirely of long filaments or
threads. This variation in morphology is known as "pleomorphism."
Some of the factors influencing pleomorphism are:
1. The composition, reaction, etc., of the _nutrient medium_ in which
the organism is growing.
2. _The atmosphere_ in which it is cultivated.
3. _The temperature_ at which it is incubated.
4. Exposure to or protection from _light_.
The various points in the anatomy morphology and physiology of bacteria
upon which stress is laid in the following pages should be studied as
closely as is possible in preparations of the micro-organisms named in
connection with each.
~ANATOMY.~
1. _Capsule_ (Fig. 85, b).--A gelatinous envelope (probably akin to
mucin in composition) surrounding each individual organism, and
preventing absolute contact between any two. In some species the capsule
(e. g., B. pneumoniae) is well marked, but it cannot be demonstrated in
all. In very well marked cases of gelatinisation of the cell wall, the
individual cells are cemented together in a coherent mass, to which the
term "zoogloea" is applied (e. g., Streptococcus mesenteroides). In
some species colouring matter or ferric oxide is stored in the capsule.
2. _Cell Wall_ (Fig. 85, c).--A protective differentiation of the
outer layer of the cell protoplasm; difficult to demonstrate, but
treatment with iodine or salt solution sometimes causes shrinkage of the
cell contents--"plasmolysis"--and so renders the cell wall apparent (_e.
g._, B. megatherium) in the manner shown in figure 85. Stained bacilli,
when examined with the polarising microscope, often show a doubly
refractile cell wall (e. g., B. tuberculosis and B. anthracis).
In some of the higher bacteria the cell wall exhibits this
differentiation to a marked degree and forms a hard sheath within which
the cell protoplasm is freely movable; and during the process of
reproduction the cell protoplasm may be extruded, leaving the empty tube
unaltered in shape.
[Illustration: FIG. 85.--Dragrammatic sketch of composite bacterium to
illustrate details of anatomical structure.]
[Illustration: FIG. 86.--Plasmolysis.]
3. _Cell Contents._--Protoplasm (mycoprotein) contains a high percentage
of nitrogen, but is said to differ from proteid in that it is not
precipitated by C_{2}H_{6}O. It is usually homogeneous in
appearance--sometimes granular--and may contain oil globules or sap
vacuoles (Fig. 85, d), chromatin granules, and even sulphur granules.
Sap vacuoles must be distinguished from spores, on the one hand, and the
vacuolated appearance due to plasmolysis, on the other.
The cell contents may sometimes be differentiated into a parietal layer,
and a central body (e. g., beggiotoa) when stained by haematoxylin.
4. _Nucleus._--This structure has not been conclusively proved to
exist, but in some bacteria chromatin particles have been observed near
the centre of the bacterial cell and denser masses of protoplasm
situated at the poles which exhibit a more marked affinity than the rest
of the cell protoplasm for aniline dyes. These latter are termed polar
granules or _Polkoerner_ (Fig. 85, e). Occasionally these aggregations
of protoplasm alter the colour of the dye they take up. They are then
known as metachromatic bodies or _Ernstschen Koerner_ (e. g., B.
diphtheriae).
5. _Flagella_ (Organs of Locomotion, Fig. 85, a).--These are
gelatinous elongations of the cell protoplasm (or more probably of the
capsule), occurring either at one pole, at both poles, or scattered
around the entire periphery. Flagella are not pseudopodia. The
possession of flagella was at one time suggested as a basis for a system
of classification, when the following types of ciliation were
differentiated (Fig. 87):
[Illustration: FIG. 87.--Types of ciliation.]
1. Polar: (a) _Monotrichous_ (a single flagellum situated at one pole;
e. g., B. pyocyaneus).
(b) _Amphitrichous_ (a single flagellum at each pole; e. g.,
Spirillum volutans).
(c) _Lophotrichous_ (a tuft or bunch of flagella situated at each
pole; e. g., B. cyanogenus).
2. Diffuse: _Peritrichous_ (flagella scattered around the entire
periphery e. g., B. typhosus).
~PHYSIOLOGY.~
~Reproduction.~--_Active Stage._--Vegetative, i. e., by the division of
cells, or "fission."
1. The cell becomes elongated and the protoplasm aggregated at opposite
poles.
2. A circular constriction of the organism takes place midway between
these aggregations, and a septum is formed in the interior of the cell
at right angles to its length.
3. The division deepens, the septum divides into two lamellae, and
finally two cells are formed.
[Illustration: FIG. 88.--Fission of cocci.]
[Illustration: FIG. 89.--Fission of bacteria.]
4. The daughter cells may remain united by the gelatinous envelope for a
variable time. Eventually they separate and themselves subdivide.
Cultures on artificial media, after growing in the same medium for some
time--i. e., when the pabulum is exhausted--show "involution forms"
(Fig. 90), well exemplified in cultures of B. pestis on agar two days
old, B. diphtheriae on potato four to six days old.
[Illustration: FIG. 90.--Involution forms.]
They are of two classes, viz.:
(a) Involution forms characterised by alterations of shape (Fig. 90).
(Not necessarily dead.)
(b) Involution forms characterised by loss of staining power. (Always
dead.)
_Resting Stage._--Spore Formation.--Conditions influencing spore
formation: In an old culture nothing may be left but spores. It used to
be supposed that spores were _always_ formed, so that the species might
not become extinct, when
(a) The supply of nutrient was exhausted.
(b) The medium became toxic from the accumulation of metabolic
products.
(c) The environment became unfavourable; e. g., change of
temperature.
This is not altogether correct; e. g., the temperature at which spores
are best formed is constant for each bacterium, but varies with
different species; again, aerobes require oxygen for sporulation, but
anaerobes will not spore in its presence.
(A) Arthrogenous: Noted only in the micrococci. One complete element
resulting from ordinary fission becomes differentiated for the purpose,
enlarges, and develops a dense cell wall. One or more of the cells in a
series may undergo this alteration.
This process is probably not real spore formation, but merely relative
increase of resistance. These so-called arthrospores have never been
observed to "germinate," nor is their resistance very marked, as they
fail to initiate new cultures, after having been exposed to a
temperature of 80 deg. C. for ten minutes.
(B) Endogenous: The cell protoplasm becomes differentiated and condensed
into a spherical or oval mass (very rarely cylindrical). After further
contraction the outer layers of the mass become still more highly
differentiated and form a distinct spore membrane, and the spore itself
is now highly refractile. It has been suggested, and apparently on good
grounds, that the spore membrane consists of two layers, the exosporium
and the endosporium. Each cell forms one spore only, usually in the
middle, occasionally at one end (some exceptions, however, are recorded;
e. g., B. inflatus). The shape of the parent cell may be unaltered, as
in the anthrax bacillus, or altered, as in the tetanus bacillus, and
these points serve as the basis for a classification of spore-bearing
bacilli, as follows:
(A) Cell body of the parent bacillus unaltered in shape (Fig. 91, a).
(B) Cell of the parent bacillus altered in shape.
1. _Clostridium_ (Fig. 91, b): Rod swollen at the centre and
attenuated at the poles; spindle shape; e. g., B. butyricus.
2. _Cuneate_ (Fig. 91, c): Rods swollen slightly at one pole and more
or less pointed at the other; wedge-shaped.