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A colloidal assembly of caseins associated with calcium phosphate in milk; changes in pH and enzyme action can destabilize it.

Cow's milk contains water, dispersed fat, casein, soluble milk proteins, lactose and mineral constituents, with smaller amounts of other substances. These constituents occupy different physical states, which makes their separation possible: the fat is present as an emulsion, much of the casein as colloidal casein micelles, and the lactose and many salts in chemical solution. Vitamins, enzymes and small nitrogenous constituents accompany these main fractions. The proportions vary between samples and are not fixed by the name cow's milk.^[ref]

First let fresh unhomogenized milk stand so that the less dense fat globules rise as cream; skimming gives a fat-enriched layer and skimmed milk beneath. This separates fractions rather than pure chemical substances: cream still contains water and other constituents. Churning cream coalesces the fat globules into butter; extraction of a separately prepared dried portion with a suitable organic solvent provides another way to isolate fat. A modern centrifugal separator accelerates the same density-based separation, but it is not needed to explain an 1864 bench separation.

Next add dilute acetic acid carefully to skimmed milk. Lowering its pH toward the isoelectric point of casein, about 4.6, produces a curd that can be collected by filtration and washed. Excess acid, retained fat, and shifts of calcium phosphate between curd and liquid limit purity. Rennet offers another route to a curd, but its enzyme-catalysed action and the mineral content of the resulting curd differ from simple acid precipitation; the two should not be treated as identical mechanisms.^[ref]

The liquid after removal of the curd is whey. Heat this fraction under suitable acidity to induce protein denaturation and aggregate much of its residual whey protein; collect the coagulum by filtration. Older descriptions commonly grouped this fraction under “albumin”, but modern milk chemistry distinguishes several proteins. Heating alone does not make every minor constituent fall into a single pure fraction.^[ref]

Concentrate the deproteinized whey and cool it to crystallize lactose. Filter and wash the crystals, then purify them by recrystallization; dissolved salts and residual organic matter remain substantially in the mother liquor. This provides a separation from most protein, not an automatic complete separation from all minerals.^[ref]

Finally use separate measured portions for water and mineral determinations. Controlled drying gives an operational total solids measurement; subtraction from the initial mass estimates the water lost. Ignition of a dried portion destroys most organic matter and leaves ash for mineral analysis. Ash is a transformed residue: it does not preserve all original salts unchanged, and some constituents can volatilize. Calcium, phosphorus, sodium, potassium and chloride are among the important mineral constituents. Skimming, curd formation, heat coagulation, lactose crystallization, drying and ashing separate or determine the principal fractions; none alone constitutes a complete pure-component analysis.

The historical name fermentation covered several transformations of organic matter: alcoholic, lactic, butyric and acetous changes, as well as some processes now classified separately. In modern biochemistry, fermentation more narrowly means energy-yielding metabolism that regenerates oxidized cofactors without requiring an external respiratory electron acceptor. The older use of the word should not be forced into that modern definition.

In alcoholic fermentation, suitable yeast converts a fermentable sugar into ethanol and carbon dioxide. For glucose, the principal net balance isA vessel with an outlet for the evolved gas and a separate receiver illustrates the change: test the carbon dioxide with limewater and identify the volatile alcohol in a separated distillate. Gas evolution alone does not prove that the liquid contains ethanol. Actual yeast cultures also make biomass and minor products; the chemical equation describes the principal conversion. In modern terms, glycolysis supplies ATP, and the reduction leading to ethanol regenerates NAD+ from NADH.

In lactic acid fermentation, an ideal homolactic balance isThe souring of suitable carbohydrate-containing materials, including milk, supplied a familiar historical example. Hydrolysis of lactose supplies monosaccharides, while acid production lowers the pH and can precipitate casein. Casein coagulation is a consequence of the changed conditions, not evidence that the protein has itself been converted into lactic acid. Some organisms instead make additional products, so not every lactic fermentation obeys only this homolactic balance.

In butyric acid fermentation, carbohydrates or lactate can give butyric acid, carbon dioxide and molecular hydrogen. An idealized balance from lactic acid isIt contrasts with the simple homolactic balance because both gaseous products and a different organic acid appear. Mixed cultures and alternative pathways can alter the product distribution; odour is neither a sufficiently specific nor a necessary laboratory test.

Acetous fermentation denotes the conversion of ethanol into acetic acid, as in vinegar production:It depends on oxygen and is now understood as an aerobic microbial oxidation, rather than a fermentation in the narrow biochemical sense. This explains why access to air matters differently here than in the main alcohol-producing stage.

Other older extensions of the word include saccharification or diastatic changes, such as the hydrolysis of starch into smaller sugars by malt enzymes, and sugar inversion, in which sucrose takes up water to form glucose and fructose:The two hexose products have the same elemental formula but different structures. Such hydrolyses prepare substrates for later metabolism; they do not by themselves constitute the alcohol-producing conversion. Viscous fermentation or mucilaginous fermentation described transformations yielding gum-like material and sometimes mannitol; it is an operational historical category without one universal balanced chemical equation. Putrefactive fermentation referred to the decomposition of nitrogen-containing organic matter, including proteolysis and subsequent reactions producing Ammonia, amines and sometimes hydrogen sulfide. It likewise has no single formula covering every mixed substrate and product.

The unifying historical interest is that small amounts of a “ferment” bring about major changes in a large amount of organic substrate. Modern explanation distinguishes organisms, enzymes and metabolic pathways. The later demonstration of fermentation in cell-free yeast juice showed that alcoholic conversion need not require intact living yeast cells in the reacting liquid; that result belongs to 1897, not to knowledge to demand of an 1866 candidate.^[ref]

Use a qualitative separation into aqueous extract, lipid-soluble material, protein-rich residue and mineral matter, and test each fraction independently. A tubercle is a lesion containing several kinds of material, not a pure chemical compound with one diagnostic formula. The question asks how to investigate its composition; no observations or numerical composition are supplied.

First record the lesion's physical appearance and take a portion for microscopy before destructive treatment. Separate the lesion from surrounding tissue as far as practicable, while recording that contamination by blood, connective tissue or adherent salts can affect the analysis. Different stages of a lesion need not have the same proportions of organic material and calcified matter. A small portion gently dried in suitable apparatus shows whether much water is lost; collecting condensation supports identification of the volatile fraction as containing water, although mass loss alone is not a chemically specific water test.

On separate portions, or by a documented extraction sequence, examine the main fractions:

Test soluble salts using a fresh portion or a suitable water extract: for example, silver nitrate can detect chloride, while barium under controlled acidic conditions can detect sulfate. A chloride test should not be performed on material already contaminated with hydrochloric acid. The results identify constituents or operational fractions, not necessarily the precise salts originally present in intact tissue; ashing can also change their chemical forms.

If the intended composition is elemental rather than proximate, investigate an isolated organic fraction separately: oxidation with heated copper(II) oxide can convert its carbon into carbon dioxide and its hydrogen into water, which are trapped and identified independently. A suitable nitrogen determination can convert organic nitrogen into Ammonia for identification in a separate receiver. Mineral carbonate and pre-existing moisture must be controlled before attributing these products to the organic material. Such tests answer a different question from identifying the particular proteins or lipids.

This approach has a historical basis. Original analyses by Robert Mortimer Glover, reproduced in an 1847 review, separately extracted material with ethanol, diethyl ether and water, then examined protein-rich residues and mineral salts. They reported variation between lesions and did not consistently find casein. This evidence supports fractionation and caution about specificity, not one universal numerical recipe for all tubercles.^[ref]

A modern conclusion must separate composition from cause. A protein-, lipid- and phosphate-containing lesion is not thereby proved to be tuberculosis. Histological interpretation and appropriate microbiological examination are different investigations. Current confirmation of Mycobacterium tuberculosis involves validated tests such as nucleic acid amplification and culture, rather than the extraction reactions above; these are modern qualifications and are not attributed to the 1866 examination.^[ref]

A preparation of milk-clotting enzymes; its action on casein differs from simple acid precipitation.