Chapter VI: Appendix: 492 (4)

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).

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).

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.

~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.

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.

_Sarcinae_, where division takes place in three planes successively, and the individuals remain attached in cubical packets of eight and its multiples.

_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.

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):

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.

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.

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.

3. _Clavate_ (Fig. 91, d): Rods swollen at one pole and cylindrical (unaltered) at the other; keyhole-shaped; e. g., B. chauvei.

4. _Capitate_ (Fig. 91, e): Rods with a spherical enlargement at one pole; drumstick-shaped; e. g., B. tetani.

The endo-spores remain within the parent cell for a variable time (in one case it is stated that germination of the spore occurs within the interior of the parent cell--"endo-germination"), but are eventually set free, as a result of the swelling up and solution of the cell membrane of the parent bacillus in the surrounding liquid, or of the rupture of that membrane. They then present the following characteristics:

1. Well-formed, dense cell membranes, which renders them extremely difficult to stain, but when once stained equally difficult to decolourise.

2. High refractility, which distinguished them from vacuoles.

3. Higher resistance than the parent organism to such lethal agents as heat, desiccation, starvation, time, etc., this resistance being due to

(a) Low water contents of plasma of the spore.

(b) Low heat-conducting power } of the spore
(c) Low permeability } membrane.

This resistance varies somewhat with the particular species--e. g., some spores may resist boiling for a few minutes--but practically all are killed if the boiling is continued for ten minutes.

~Germination.~--When transplanted to suitable media and placed under favourable conditions, the spores germinate, usually within twenty-four to thirty-six hours, and successively undergo the following changes which may be followed in hanging-drop cultures on a warm stage:

1. Swell up slowly and enlarge, through the absorption of water.

2. Lose their refrangibility.

3. At this stage one of three processes (but the particular process is always constant for the same species) may be observed:

(a) The spore grows out into the new bacillus without discarding the spore membrane (which in this case now becomes the cell membrane); _e. g._, B. leptosporus.

(b) It loses its spore membrane by solution; e. g., B. anthracis.

(c) It loses its spore membrane by rupture.

In this process the rupture may be either polar (at one pole only _e. g._, B. butyricus), or bipolar (e. g., B. sessile), or equatorial; (e. g., B. subtilis).

In those cases where the spore membrane is discarded the cell membrane of the new bacillus may either be formed from--

(a) The inner layer of the spore membrane, which has undergone a preliminary splitting into parietal and visceral layers; e. g., B. butyricus.

(b) The outer layers of the cell protoplasm, which become differentiated for that purpose; e. g., B. megatherium.

The new bacillus now increases in size, elongates, and takes on a vegetative growth--i. e., undergoes fission--the bacilli resulting from which may in their turn give rise to spores.

~Food Stuffs.~--1. _Organic Foods._--

(a) The pure parasites (e. g., B. leprae) will not live outside the living body.

(b) Both saprophytic and facultative parasitic bacteria agree in requiring non-concentrated food.

(c) The facultative parasites need highly organised foods; e. g., proteids or other sources of nitrogen and carbon, and salts.

(d) The saprophytic bacteria are more easily cultivated; e. g.,

1. Some bacteria will grow in almost pure distilled water.

2. Some bacteria will grow in pure solutions of the carbohydrates.

3. _Water_ is absolutely essential to the _growth_ of bacteria.

Food of a definite reaction is needed for the growth of bacteria. As a general rule growth is most active in media which react slightly acid to phenolphthalein--that is, neutral or faintly alkaline to litmus. Mould growth, on the other hand, is most vigourous in media that are strongly acid to phenolphthalein.

~Environment.~--The influence of physical agents upon bacterial life and growth is strongly marked.

1. _Atmosphere._--The presence of _oxygen_ is necessary for the growth of some bacteria, and death follows when the supply is cut off. Such organisms are termed _obligate aerobes_.

Some bacteria appear to thrive equally well whether supplied with or deprived of oxygen. These are termed _facultative anaerobes_.

A third class will only live and multiply when the access of free oxygen is completely excluded. These are termed _obligate anaerobes_.

2. _Temperature._--Practically no bacterial growth occurs below 5 deg. C, and very little above 40 deg. C. 30 deg. C. to 37 deg. C is the most favorable for the large majority of micro-organisms.

The maximum and minimum temperatures at which growth takes place, as well as the optimum, are fairly constant for each bacterium.

Bacteria have been classified, according to their optimum temperature, into--

MIN. OPT. MAX.

1. Psychrophilic bacteria (chiefly water organisms) 0 deg. C. 15 deg. C. 30 deg. C. 2. Mesophilic bacteria (includes pathogenic bacteria) 15 deg. C. 37 deg. C. 45 deg. C. 3. Thermophilic bacteria 45 deg. C. 55 deg. C. 70 deg. C.

The thermal death-point of an organism is another biological constant; and is that temperature which causes the death of the vegetative forms when the exposure is continued for a period of ten minutes (see pages 298-301).

3. _Light._--Many organisms are indifferent to the presence of light. On the other hand, light frequently impedes growth, and alters to a greater or lesser extent the biochemical characters of the organisms--e. g., chromogenicity or power of liquefaction. Pathogenic bacteria undergo a progressive loss of virulence when cultivated in the presence of light.

4. _Movements._--Movements, if slight and simply of a flowing character, do not appear to injuriously affect the growth of bacteria; but violent agitation, such as shaking, absolutely kills them.

A condition of perfect rest would seem to be that most conducive to bacterial growth.

~The Metabolic Products of Bacteria.~--_Pigment Production._--Many micro-organisms produce one or more vivid pigments--yellow, orange, red, violet, fluorescent, etc.--during the course of their life and growth. The colouring matter usually exists as an intercellular excrementitious substance. Occasionally, however, it appears to be stored actually within the bodies of the bacteria. The chromogenic bacteria are therefore classified, in accordance with the final destination of the colouring matter they elaborate, into--

_Chromoparous_ Bacteria: in which the pigment is diffused out upon and into the surrounding medium.

_Chromophorous_ Bacteria: in which the pigment is stored in the cell protoplasm of the organism.

_Parachromophorous_ Bacteria: in which the pigment is stored in the cell wall of the organism.

Different species of chromogenic bacteria differ in their requirements as to environment, for the production of their characteristic pigments; e. g., some need oxygen, light, or high temperature; others again favor the converse of these conditions.

_Light Production._--Some bacteria, and usually those originally derived from water, whether fresh or salt, exhibit marked phosphorescence when cultivated under suitable conditions. These are classed as "photogenic."

_Enzyme Production._--Many bacteria produce soluble ferments or enzymes during the course of their growth, as evidenced by the liquefaction of gelatine, the clotting of milk, etc. These ferments may belong to either of the following well-recognised classes: proteolytic, diastatic, invertin, rennet.

_Toxin Production._--A large number, especially of the pathogenic bacteria, elaborate or secrete poisonous substances concerning which but little exact knowledge is available, although many would appear to be enzymic in their action.

These toxins are usually differentiated into--

_Extracellular_ (or Soluble) Toxins: those which are diffused into, and held in solution by, the surrounding medium.

_Intracellular_ (or Inseparate) Toxins: those which are so closely bound up with the cell protoplasm of the bacteria elaborating them that up to the present time no means has been devised for their separation or extraction.

_End-products of Metabolism._--Under this heading are included--

Organic Acids (e. g., lactic, butyric, etc.).

Alkalies (e. g., ammonia).

Aromatic Compounds (e. g., indol, phenol).

Reducing Substances (e. g., those reducing nitrates to nitrites).

Gases (e. g., sulphuretted hydrogen, carbon dioxide, etc.).

And while the discussion of their formation, etc., is beyond the scope of a laboratory handbook, the methods in use for their detection and separation come into the ordinary routine work and will therefore be described (_vide_ page 276 _et seq._).

X. NUTRIENT MEDIA.

In order that the life and growth of bacteria may be accurately observed in the laboratory, it is necessary--

1. To _isolate_ individual members of the different varieties of micro-organisms.

2. To _cultivate_ organisms, thus isolated, apart from other associated or contaminating bacteria--i. e., in _pure culture_.

For the successful achievement of these objects it is necessary to provide nutriment in a form suited to the needs of the particular bacterium or bacteria under observation, and in a general way it may be said that the nutrient materials should approximate as closely as possible, in composition and character, to the natural pabulum of the organism.

The general requirements of bacteria as to their food-supply have already been indicated (page 142) and many combinations of proteid and of carbohydrate have been devised, from time to time, on those lines. These, together with various vegetable tissues, physiological or pathological fluid secretions, etc., are collectively spoken of as _nutrient media_ or _culture media_.

The greater number of these media are primarily _fluid_, but, on account of the rapidity with which bacterial growth diffuses itself through a liquid, it is impossible to study therein the characteristics of individual organisms. Many such media are, therefore, subsequently rendered solid by the addition of substances like gelatine or agar, in varying proportions, the proportions of such added material being generally mentioned when referring to the media; e. g., 10 per cent. gelatine, 2 per cent. agar. Gelatine is employed for the solidification of those media it is intended to use in the cultivation of bacteria at the room temperature or in the "cold" incubator. In the percentages usually employed, gelatine media become fluid at 25 deg. C.; higher percentages remain solid at somewhat higher temperatures, but the difficulty of filtering strong solutions of gelatine militates against their general use.

Media, on the other hand which have been solidified by the addition of agar, only become liquid when exposed to 90 deg. C. for about ten minutes, and again solidify when the temperature falls to 40 deg. C.

When it becomes necessary to render these media fluid, heat is applied, upon the withdrawal of which they again assume their solid condition. Such media should be referred to as _liquefiable media_; in point of fact, however, they are usually grouped together with the solid media.

NOTE.--It must here be stated that the designation 10 per
cent. gelatine or 2 per cent. agar refers only to the
quantity of those substances actually added in the process
of manufacture, and _not_ to the percentage of gelatine or
agar, as the case may be, present in the finished medium;
the explanation being that the commercial products employed
contain a large proportion of insoluble material which is
separated off by filtration during the preparation of the
liquefiable media.

Other media, again--e. g., potato, coagulated blood-serum, etc.--cannot be again liquefied by physical means, and these are spoken of as _solid_ media.

The following pages detail the method of preparing the various nutrient media, in ordinary use (see also Chapter XI), those which are only occasionally required for more highly specialised work are grouped together in Chapter XII. It must be premised that scrupulous cleanliness is to be observed with regard to all apparatus, vessels, funnels, etc., employed in the preparation of media; although in the preliminary stages of the preparation of most media absolute sterility of the apparatus used is not essential.

MEAT EXTRACT.

A watery solution of the extractives, etc., of lean meat (usually beef) forms the basis of several nutrient media. This solution is termed "meat extract" and it has been determined empirically that its preparation shall be carried out by extracting half a kilo of moist meat with one litre of water. For many purposes, however, it is more convenient to have a more concentrated extract; one kilo of meat should therefore be extracted with one litre of water, to form "Double Strength" meat extract.

It was customary at one time, and is even now in some laboratories to use either "shin of beef" or "beef-steak"--both contain muscle sugar which often needs to be removed before the nutrient medium can be completed. Heart muscle (bullock's heart or sheep's heart) is much to be preferred and from the point of economy, ease and cleanliness of manipulation, and extractive value, the imported frozen bullock's hearts provide the best extract.

Meat extract (Fleischwasser) is prepared as follows:

1. Measure 1000 c.c. of distilled water into a large flask (or glass beaker, or enamelled iron pot) and add 1000 grammes (roughly, 2-1/2 pounds) of fresh lean meat--e. g., bullock's heart--finely minced in a mincing machine.

2. Heat the mixture gently in a water-bath, taking care that the temperature of the contents of the flask does not exceed 40 deg. C. for the first twenty minutes. (This dissolves out the soluble proteids, extractives, salts, etc.)

3. Now raise the temperature of the mixture to the boiling-point, and maintain at this temperature for ten minutes. (This precipitates some of the albumins, the haemoglobin, etc., from the solution.)

4. Strain the mixture through sterile butter muslin or a perforated porcelain funnel, then filter the liquid through Swedish filter paper into a sterile "normal" litre flask, and when cold make up to 1000 c.c. by the addition of distilled water--to replace the loss from evaporation.

5. If not needed at once, sterilise the meat extract in bulk in the steam steriliser for twenty minutes on each of three consecutive days.

Calf, sheep, or chicken flesh is occasionally substituted for the beef; or the meat extract may be prepared from animal viscera, such as brain, spleen, liver, or kidneys.

NOTE.--As an alternative method, 5 c.c. of Brand's meat
juice or 3 grammes of Wyeth's beef juice, or 10 grammes
Liebig's extract of meat (Lemco) may be dissolved in 1000
c.c. distilled water, and heated and filtered as above to
form ordinary or single strength meat extract.

Media, prepared from such meat extracts are, however,
eminently unsatisfactory when used for the cultivation of
the more highly parasitic bacteria; although when working in
tropical and subtropical regions their use is well-nigh
compulsory.

~Reaction of Meat Extract.~--Meat extract thus prepared is acid in its reaction, owing to the presence of acid phosphates of potassium and sodium, weak acids of the glycolic series, and organic compounds in which the acid character predominates. Owing to the nature of the substances from which it derives its reaction, the total acidity of meat extract can only be estimated accurately when the solution is at the boiling-point.

Moreover, it has been observed that prolonged boiling (such as is involved in the preparation of nutrient media) causes it to undergo hydrolytic changes which increase its acidity, and ~the meat extract only becomes stable in this respect after it has been maintained at the boiling-point for forty-five minutes~.

Although meat extract always reacts acid to phenolphthalein, it occasionally reacts neutral or even alkaline to litmus; and again, meat extract that has been rendered exactly neutral to litmus still reacts acid to phenolphthalein. This peculiar behaviour depends upon two factors:

1. Litmus is insensitive to many weak organic acids the presence of which is readily indicated by phenolphthalein.

2. Dibasic sodium phosphate which is formed during the process of neutralisation is a salt which reacts alkaline to litmus, but neutral to phenolphthalein. In order, therefore, to obtain an accurate estimation of the reaction of any given sample of meat extract, it is essential that--

1. The meat extract be previously exposed to a temperature of 100 deg. C. for forty-five minutes.

2. The estimation be performed at the boiling-point.

3. Phenolphthalein be used as the indicator.

The estimation is carried out by means of titration experiments against standard solutions of caustic soda, in the following manner:

_Method of Estimating the Reaction._--

_Apparatus Required_: _Solutions Required_:

1. 25 c.c. burette graduated 1. 10N NaOH, accurately in tenths of a centimetre. standardised.

2. 1 c.c. pipette graduated in 2. n/1 NaOH, accurately hundredths, and provided standardised with rubber tube, pinch-cock, and delivery nozzle.

3. 25 c.c. measure (cylinder or 3. n/10 NaOH, accurately pipette, calibrated for standardised. 98 deg. C.--_not_ 15 deg. C).

4. Several 60 c.c. conical 4. 0.5 per cent. solution of beakers or Erlenmeyer phenolphthalein in 50 per flasks. cent. alcohol.

5. White porcelain evaporating basin, filled with boiling water and arranged over a gas flame as a water-bath.

6. Bohemian glass flask, fitted as a wash-bottle, and filled with distilled water, which is kept boiling on a tripod stand.

METHOD.--Arrange the apparatus as indicated in figure 97.

(A) 1. Fill the burette with n/10 NaOH.

2. Fill the pipette with n/1 NaOH.

3. Measure 25 c.c. of the meat extract (previously heated in the steamer at 100 deg. C. for forty-five minutes) into one of the beakers by means of the measure; rinse out the measure with a very small quantity of boiling distilled water from the wash-bottle, and then add this rinse water to the meat extract already in the beaker.

4. Run in about 0.5 c.c. of the phenolphthalein solution and immerse the beaker in the water-bath, and raise to the boil.

5. To the medium in the beaker run in n/10 NaOH cautiously from the burette until the end-point is reached, as indicated by the development of a pinkish tinge, shown in figure 98 (b). Note the amount of decinormal soda solution used in the process.

NOTE.--Just before the end-point is reached, a very slight
opalescence may be noted in the fluid, due to the
precipitation of dibasic phosphates. After the true
end-point is reached, the further addition of about 0.5 c.c.
of the decinormal soda solution will produce a deep magenta
colour (Fig. 98, c), which is the so-called "end-point" of
the American Committee of Bacteriologists.

(B) Perform a "control" titration (occasionally two controls may be necessary), as follows:

1. Measure 25 c.c. of the meat extract into one of the beakers, wash out the measure with boiling water, and add the phenolphthalein as in the first estimation.

2. Run in n/1 NaOH from the pipette, just short of the equivalent of the amount of _deci_-normal soda solution required to neutralise the 25 c.c. of medium. (For example, if in the first estimation 5 c.c. of n/10 NaOH were required to render 25 c.c. of medium neutral to phenolphthalein, only add 0.48 c.c. of n/1 NaOH.) Immerse the beaker in the water-bath.

3. Complete the titration by the aid of the n/10 NaOH.

4. Note the amount of n/10 NaOH solution required to complete the titration, and add it to the equivalent of the n/1 NaOH solution previously run in. Take the total as the correct estimation.

_Method of Expressing the Reaction._--

The reaction or _titre_ of meat extract, medium, or any solution estimated in the foregoing manner, is most conveniently expressed by indicating the number of cubic centimetres of normal alkali (or normal acid) that would be required to render _one litre_ of the solution exactly neutral to phenolphthalein.

The sign + (plus) is prefixed to this number if the original solution reacts acid, and the sign - (minus) if it reacts alkaline.

For example, "meat extract + 10," indicates a sample of meat extract which reacts acid to phenolphthalein, and would require the addition of 10 c.c. of _normal_ NaOH per litre, to neutralise it.

NOTE.--Such a solution would probably react alkaline to
litmus.

Conversely, if as the result of our titration experiments we find that 25 c.c. of meat extract require the addition of 5 c.c. n/10 NaOH to neutralise, then 1000 c.c. of meat extract will require the addition of 200 c.c. n/10 NaOH = 20 c.c. n/1 NaOH.

And this last figure, 20, preceded by the sign + (i. e., +20), to signify that it is acid, indicates the reaction of the meat extract.

NOTE.--The standard soda solutions should be prepared by
accurate measuring operations, controlled by titrations,
from a stock solution of 10N NaOH, which should be very
carefully standardised. If a large supply is made or the
consumption is small this stock solution must be kept in an
aspirator bottle to which air can only gain access after it
has been dried and rendered free from CO_{2}. This may be
done by first leading it over H_{2}SO_{4} and soda lime, or
soda lime alone, by some such arrangement as is shown in
figure 99, which also shows a constant burette arrangement
for the delivery of small measured quantities of the
dekanormal soda solution.

STANDARDISATION OF MEDIA.

Differences in the reaction of the medium in which it is grown will provoke not only differences in the rate of growth of any given bacterium, but also well-marked differences in its cultural and morphological characters; and nearly every organism will be found to affect a definite "optimum reaction"--a point to be carefully determined for each. For most bacteria, however, the "optimum" usually approximates fairly closely to +10; and as experiment has shown that this reaction is the most generally useful for routine laboratory work, it is the one which may be adopted as the standard for all nutrient media derived from meat extract.

Briefly, the method of standardising a litre of media to +10 consists in subtracting 10 from the initial _titre_ of the medium mass; the remainder indicates the number of cubic centimetres of normal soda solution that must be added to the medium, per litre, to render the reaction +10.

~Standardising Nutrient Bouillon.~--For example, 1000 c.c. bouillon are prepared; at the first titration it is found

1. 25 c.c. require the addition of 5.50 c.c. n/10 NaOH to neutralise.

Two controls give the following results:

2. 25 c.c. require the addition of 5.70 c.c. n/10 NaOH to neutralise.

3. 25 c.c. require the addition of 5.60 c.c. n/10 NaOH to neutralise.

Averaging these two controls, 25 c.c. require the addition of 5.65 c.c. n/10 NaOH to neutralise, and therefore 1000 c.c. require the addition of 226 c.c. n/10 NaOH, or 22.60 c.c. n/1 NaOH, or 2.26 c.c. n/10 NaOH.

Initial _titre_ of the bouillon = +22.6, and as such requires the addition of (22.6 c.c. - 10 c.c.) = 12.6 c.c. of n/1 NaOH per litre to leave its finished reaction +10.

But the three titrations, each on 25 c.c. of medium, have reduced the original bulk of bouillon to (1000 - 75 c.c.) = 925 c.c. The amount of n/1 NaOH required to render the reaction of this quantity of medium +10 may be deduced thus:

1000 c.c.:925 c.c.::12.6 c.c.:x.

Then x = 11.65 c.c. n/1 NaOH.

Whenever possible, however, the required reaction is produced by the addition of dekanormal soda solution, on account of the minute increase it causes in the bulk, and the consequent insignificant disturbance of the percentage composition of the medium. By means of a pipette graduated to 0.01 c.c. it is possible to deliver very small quantities; but if the calculated amount runs into thousandth parts of a cubic centimetre, these are replaced by corresponding quantities of normal or even decinormal soda.

In the above example it is necessary to add 11.65 c.c. normal NaOH or its equivalent, 1.165 c.c. dekanormal NaOH. The first being too bulky a quantity, and the second inconveniently small for exact measurement, the total weight of soda is obtained by substituting 1.16 c.c. dekanormal soda solution, and either 0.05 c.c. of normal soda solution or 0.5 c.c. of decinormal soda solution.

~Standardising Nutrient Agar and Gelatine.~--The method of standardising agar and gelatine is precisely similar to that described under bouillon.

THE FILTRATION OF MEDIA.

~Fluid media~ are usually filtered through stout Swedish filter paper (occasionally through a porcelain filter candle), and in order to accelerate the rate of filtration the filter paper should be folded in that form which is known as the "physiological filter," not in the ordinary "quadrant" shape, as by this means a large surface is available for filtration and a smaller area in contact with the glass funnel supporting it.

To fold the filter proceed thus:

1. Take a circular piece of filter paper and fold it exactly through its centre to form a semicircle (Fig. 100, a).

2. Fold the semicircle exactly in half to form a quadrant; make the crease 2, distinct by running the thumbnail along it, then open the filter out to a semicircle again.

3. Fold each end of the semicircle in to the centre and so form another quadrant; smooth down the two new creases 3 and 3a, thus formed and again open out to a semicircle.

4. The semicircle now appears as in figure 100, a, the dark lines indicating the creases already formed.

5. Fold the point 1 over to the point 3, and 1a to 3a, to form the creases 4 and 4a, indicated in the diagram by the light lines. Fold point 1 over to 3a, and 1a to 3, to form the creases 5 and 5a.

6. Thus far the creases have all been made on the same side of the paper. Now subdivide each of the eight sectors by a crease through its centre on the opposite side of the paper, indicated by the faint broken lines in the diagram. Fold up the filter gradually as each crease is made, and when finished the filter has assumed the shape of a wedge, as in figure 100, b.

When opened out the filter assumes the shape represented in figure 100, c.

The folded filter is next placed inside a glass funnel supported on a retort stand, and moistened with hot distilled water before the filtration of the medium is commenced.

~Liquefiable solid media~ are filtered through a specially made filter paper--"papier Chardin"--which is sold in boxes of twenty-five ready-folded filters.

Gelatine, when properly made, filters through this paper as quickly as bouillon does through the Swedish filter paper, and does _not_ require the use of the hot-water funnel.

Agar, likewise, if properly made, filters readily, although not at so rapid a rate as gelatine. If badly "egged," and also during the winter months, it is necessary to surround the glass funnel, in which the filtration of the agar is carried on, by a hot-water jacket. This is done by placing the glass funnel inside a double-walled copper funnel--the space between the walls being filled with water at about 90 deg. C.--and supporting the latter on a ring gas burner fixed to a retort stand (Fig. 101). The gas is lighted and the water jacket maintained at a high temperature until filtration is completed. If the steam steriliser of the laboratory is sufficiently large, it is sometimes more convenient to place the flask and filtering funnel bodily inside, close the steriliser and allow filtration to proceed in an atmosphere of live steam, than to use the gas ring and hot-water funnel.

STORING MEDIA IN BULK.

After filtration fill the medium into sterile litre flasks with cotton-wool plugs and sterilise in the steamer for twenty minutes on each of three consecutive days. After the third sterilisation, and when the flasks and contents are cool, cut off the top of the cotton-wool plug square with the mouth of the flask; push the plug a short distance down into the neck of the flask and fill in with melted paraffin wax to the level of the mouth. When the wax has set the flasks are stored in a cool dark cupboard for future use.

This plan is not absolutely satisfactory, although very generally employed on occasion, and it is preferable to fill the medium into long-necked flint glass bottles (the quart size, holding nearly 1000 c.c., such as those in which Pasteurised milk is retailed) and to close the neck of the bottle by a special rubber cap.[3] This cap is made of soft rubber, the lower part, dome-shaped with thin walls, being slipped over the neck of the bottle (Fig. 102, a). The upper part is solid, but with a sharp clean-cut (made with a cataract or tenotomy knife) running completely through its axis from the centre of the disc to the top of the dome. During sterilisation the air in the neck of the bottle, expanded by the heat, is driven out through the valvular aperture in the solid portion of the stopper. On removing the bottle from the steam chamber, the liquid contracts as it cools, and the pressure of the external air drives the solid piece of rubber down into the neck of the bottle, and forces together the lips of the slit (Fig. 102, b). Thus sealed, the bottle will preserve its contents sterile for an indefinite period without loss from evaporation.

TUBING NUTRIENT MEDIA.

After the final filtration, the nutrient medium is usually "tubed"--_i. e._, filled into sterile tubes in definite measured quantities, usually 10 c.c. This process is sometimes carried out by means of a large separator funnel fitted with a "three-way" tap which communicates with a small graduated tube (capacity 20 c.c. and graduated in cubic centimetres) attached to the side. The shape of this piece of apparatus, known as Treskow's funnel, renders it particularly liable to damage. It is better, therefore, to arrange a less expensive piece of apparatus which will serve the purpose equally well (Fig. 103).

A Geissler's three-way stop-cock has the tube on one side of the tap ground obliquely at its extremity, and the tube on the opposite side cut off within 3 cm. of the tap. The short tube is connected by means of a perforated rubber cork with a 10 cm. length of stout glass tubing (1.5 cm. bore). The third channel of the three-way tap is connected, by means of rubber tubing, with the nozzle of an ordinary separator funnel. Finally, the receiving cylinder above the three-way tap is graduated in cubic centimetres up to 20, by pouring into it measured quantities of water and marking the various levels on the outside with a writing diamond.

Fluid media containing carbohydrates are filled into fermentation tubes (_vide_ Fig. 21); or into ordinary media tubes which already have smaller tubes, inverted, inside them (Fig. 104), to collect the products of growth of gas-forming bacteria. When first filled, the small tubes float on the surface of the medium after the first sterilisation nearly all the air is replaced by the medium, and after the final sterilisation the gas tubes will be submerged and completely filled with the medium.

~Storing "Tubed" Media.~--Media after being tubed are best stored by packing, in the vertical position, in oblong boxes having an internal measurement of 37 cm. long by 12 cm. wide by 10 cm. deep. Each box (Fig. 105) has a movable partition formed by the vertical face of a weighted triangular block of wood, sliding free on the bottom (Fig. 105, A); or by a flat piece of wood sliding in a metal groove in the bottom of the box, which can be fixed at any spot by tightening the thumbscrew of a brass guide rod which transfixes the partition (Fig. 105, B). The front of the box is provided with a handle and a celluloid label for the name of the contained medium. These boxes are arranged upon shelves in a dark cupboard--or preferably an iron safe--which should be rendered as nearly air-tight as possible, and should have the words "media stores" painted on its doors.

FOOTNOTES:

[3] This rubber cap has been made for me by the Holborn Surgical Instrument Co., Thavies Inn, London, W. C.

XI. CULTURE MEDIA.

ORDINARY OR STOCK MEDIA.

~Nutrient Bouillon.~--

1. Measure out double strength meat extract, 500 c.c., into a litre flask and add 300 c.c. distilled water.

2. Weigh out Witte's peptone, 10 grammes (= 1 per cent.), salt, 5 grammes (= 0.5 per cent.), and mix into a smooth paste with 200 c.c. of distilled water previously heated to 60 deg. C. (Be careful to leave no unbroken globular masses of peptone.)

3. Add the peptone emulsion to the meat extract in the flask and heat in the steamer for forty-five minutes (to completely dissolve the peptone, and to render the acidity of the meat extract stable).

4. Estimate the reaction of the medium; control the result; render the reaction of the finished medium +10 (_vide_ page 155).

5. Heat for half an hour in the steamer at 100 deg. C. (to complete the precipitation of the phosphates, etc.).

6. Filter through Swedish filter paper into a sterile flask.

7. Fill into sterile tubes (10 c.c. in each tube).

8. Sterilise in the steamer for twenty minutes on each of three consecutive days--i. e., by the discontinuous method (_vide_ page 35).

NOTE.--As an alternative method when neither fresh nor
frozen meat is available nutrient bouillon may be prepared
from a commercial meat extract, as follows:

~Lemco Broth.~--

1. Measure out 250 c.c. distilled water into a litre flask.

2. Weigh out 10 grammes Liebig's Lemco Meat Extract on a
piece of clean filter paper and add to the water in the
flask. Shake the flask well to make an even emulsion of the
meat extract.

3. Weigh out Witte's peptone (10 grammes), salt (5 grammes).
Mix into smooth paste with 100 c.c. distilled water
previously heated to 60 deg. C.

4. Add the peptone salt emulsion to the meat extract
emulsion in the flask and add 650 c.c. distilled water. Heat
in the steamer for forty-five minutes.

5. Standardise the medium and complete as for nutrient
bouillon.

~Nutrient Gelatine.~--

1. Weigh a 2-litre flask on a trip balance (Fig. 106) and note the weight, or counterpoise carefully.

An extremely useful counterpoise is a small sheet-brass cylinder about 38 mm. high and 38 mm. in diameter, with a funnel-shaped top and provided with a side tube by which its contents, fine "dust" shot, may be emptied out (Fig. 107).

2. Measure out double strength meat extract, 500 c.c., into the "tared" flask.

3. Weigh out and mix 10 grammes of peptone, 5 grammes of salt, and make into a thick paste with 150 c.c. distilled water; then add the emulsion to the meat extract in the flask; also add 100 grammes sheet gelatine cut into small pieces; place the flask in the water-bath and raise to the boil.

4. Arrange a 5-litre tin can (with copper bottom, such as is used in the preparation of distilled water) by the side of the water bath, fill the can with boiling water and place a lighted Bunsen burner under it. Fit a long safety tube to the neck of the can and also a delivery tube, bent twice at right angles; adjust the tube to reach to the bottom of the interior of the flask containing the gelatine, etc. (Fig. 108).

5. Keep the water in the steam can vigourously boiling, and so steam at 100 deg. C, bubbling through the medium mass, for ten minutes, by which time complete solution of the gelatine is effected. A certain amount of steam will condense as water in the medium flask during this process--hence the necessity for the use of double strength meat extract--but if the water bath is kept boiling this condensation will not exceed 100 c.c.

6. Weigh the flask and its contents; then (1115[4] grammes + weight of the flask) minus (weight of the flask and its contents) equals the weight of water required to make up the bulk to 1 litre. The addition of the requisite quantity of water is carried out as follows:

In one pan of the trip balance place the counterpoise of the tared flask (or its equivalent in weights) together with the weights making up the _calculated medium weight_. In the opposite pan place the flask containing the medium mass. Now add boiling distilled water from a wash bottle until the two pans are exactly balanced.