= Solution
\b[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:
* The <water> extract contains soluble salts and some soluble organic material. Test its reaction with <litmus>; heating a suitable clear portion can disclose coagulable <protein>. Acidification and heating must be controlled because changes of <pH> and high salt content can alter coagulation. The absence of a heat coagulum does not prove the absence of every <protein>.
* An <ethanol> or <diethyl ether> extraction can remove <lipids> and other solvent-soluble material. Evaporating a separated solvent extract leaves material whose solubility, oily or crystalline appearance, and behaviour on heating help characterize it. Hydrolysable fats can undergo <saponification>; <cholesterol> belongs to the unsaponifiable fraction. A greasy residue alone does not establish one particular <lipid>.
* Examine the largely insoluble organic residue for <protein>. A controlled alkaline copper reaction gives the violet <biuret test> with sufficient <peptide bonds>; the yellow <xanthoproteic reaction> with <nitric acid> supplies corroborating evidence for suitable aromatic residues. Neither colour establishes that all the material is specifically <albumin>, <casein> or a hypothetical unique “tuberculin” substance. Record the reaction conditions and use a reagent blank and known positive material.
* Ignite a separate portion carefully to remove combustible organic material, then examine the <ash>. Dissolve suitable portions in a noninterfering <chemical acid>. <Calcium> can be recognized by <oxalate> precipitation under appropriate conditions; <phosphate> by an established phosphate test such as the yellow <ammonium phosphomolybdate> reaction. <Carbonate> can be recognized by acid-induced <carbon dioxide> evolution and a separate <limewater> test. These are separate aliquots: acid treatment used to dissolve the <ash> can destroy evidence for the original <carbonate>.
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.<Analyses of tubercular material by Robert Mortimer Glover>[1846 book passages reproduced in Dr. Glover on Scrofula, printed pp. 516–517; original analysis cited as book p. 73]{ref}
A modern conclusion must separate \b[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.<Clinical and Laboratory Diagnosis for Tuberculosis by CDC>[Bacteriologic examination, nucleic acid amplification and culture sections]{ref}
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