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.
- 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.[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]
Clinical and Laboratory Diagnosis for Tuberculosis by CDC. Bacteriologic examination, nucleic acid amplification and culture sections
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