An Exchange in Locard’s Own Words (Part 5)

Translated by Kathleen Brahney
Commissioned by McCrone Associates, Inc.

Edmond Locard
Doctor of Medicine, Professor of Law, Director of the Lyon Laboratory of Police Techniques,
Vice-President of the International Academy of Criminology

Manual of Police Techniques
Third Edition, Completely Revised and Augmented.
Paris: Payot, 16 Boulevard St. Germain, 1939

Part V—Body-fluid Stains

Chapter IV


Semen found on bed linens takes the shape of geographic maps; on velvet or plush material, it forms dusty or scaly trails; on skin it takes the form of shiny crusts or scales.  When the convicting evidence bearing traces of semen must be transported, one must take every precaution against shaking, which could destroy the stains entirely.  Analysis can be based on the crystallographic method or by analyzing the figurative elements.

  1. Florence’s Method:Prepare the following reactive, first dissolving the iodide in the least possible quantity of water:1.56 g. of potassium iodide [KI]
    2.54 g. of washed metalloid iodine [I2]
    30 g. of distilled water [H2O]The reactive will keep for a long time.Next, wet a part of the stain with a drop of distilled water and add a few drops of the reactive.  If there is semen present, mahogany brown crystals will appear;  the form of the crystals is variable, sometimes in blades, sometimes needle-shaped, sometimes as twin crystals.  The drawback of the reaction is that it also occurs with liquid from the prostate gland and with saliva, and it does not take place when the semen is spoiled (putrified).  But the test is very sensitive and easy to do.
  2. Barberio’s Method:First one prepares a saturated solution of picric acid in water.  Filter it until there are no crystals of picric acid deposited, which could lead to confusion.  It is preferable to use a glycerinated solution (Cevidalli, Stockis).  The operation is carried out as in Florence’s method.  The yellow crystals that are obtained in the presence of semen have the drawback of also being produced in the presence of organic substances such as orange juice and pyridine.  The specificity of Barberio’s reaction is thus inferior to that of Florence.
  3. Analysis of spermatozoa:One must avoid scratching the stain; doing so would crush the semenatozoids.  The best way to take a sample is to remove a thread of the cloth and color it with the following reactive (Corin and Stockis):0.50 g of erythrosine
    100 cc of ammoniaThis reactive is quite unstable and should be prepared as needed.  De Rechter obtained a more stable reactive by mixing equal parts of erythrosine in a solution saturated with methyl alcohol, and liquid ammonia.One then dissociates it on the slide using a drop of the following solution:0.50 gr. of methylene blue
    100 cc. of distilled waterThe spermatozoa will be red, and have their characteristic shape — spoon-shaped with a neck and a tail.One can replace the erythrosine-ammonia with crocein [a red or orange azo dye] or with this solution:1 g. iodine
    4 g. potassium iodide
    100 g. distilled water

    On wood, one can likewise detach a small fiber and color it with the erythrosine-ammonia and with the methylene blue, but without dissociating it.  If one is using a detached flake, coloring it with crocein gives good results.It is well understood that the discovery of spermatozoa yields an affirmative and absolutely certain response.  But the absence of spermatozoa does not indicate a negative certainty because the cells may have decomposed if the stain is spoiled (putrified) or too old.  The reactions of Florence and Barberio therefore allow one to draw conclusions with a high degree of probability.

  4. Fixing spermatozoa with silver nitrate (Pellissier and Cordonnier, Annals of Legal Medicine, Paris, Jan. 1921):The principle of this method is to color the sperm without coloring the background.  One takes a quarter of a square centimeter of the cloth (bearing the stain) and immerses it in a 5% solution of commercial formalin for 24 hours.  Then rinse it and place the square in a mixture of equal parts of 95% alcohol and pyridine; leave it for an hour and then rinse it again.Leave the little square in a bath of 3% silver nitrate for 6 hours in a heater set at 50 degrees (for wood, one would leave it there for 12 – 24 hours.)  Then wash it in distilled water and reduce it for 6 or 7 hours in the following solution:100 [parts] water
    5 parts formalin
    4 parts pyrogallic acid
    8 parts pyridineOr in this solution:
    100 [parts] water
    1.6 parts hydroquinone
    10 parts sulfite of soda
    8 parts pyridineRinse it in running water for a half hour; then leave it in distilled water to which a few drops of formalin have been added.  To mount the specimen, dissociate a small bit on a slide in a drop of distilled water; evaporate the water on a heating plate without cooking the preparation.  Mount it on a piece of Canadian balsam.The spermatozoa will be deposited in little nests at the intersection of two threads or in the form of bouquets stuck to the fibers with a glutinous substance which reduces lightly to silver.
  5. Examination with Ultraviolet Rays:If one places a piece of cloth bearing semen stains under a Wood lamp or a Gallois lamp equipped with a quartz filter, the ultraviolet rays will give a mauve fluorescence on the stain, a fluorescence that does not occur with other organic stains (leucorrhea or gonorrheal pus).  This method does not suffice in making a diagnosis, but it does allow for the rapid identification of stains for which one can then carry out an analysis, and it eliminates other stains.  It is a way of clearing away the rubble and is very useful when there is a large amount of cloth to examine.Andre Husson (“On the Detection and Identification of semen stains in Criminalistics, Using the Wood Light.”  International Review of Criminalistics, 1934, no. 7), who has specialized in the study of semen identification using physics, has drawn the following conclusions:a)  The fluorescence of semen using the Wood light (composed of radiations 2.967 at 3.906 Ao) is bluish-white, intense and can be brought out by minimal traces of the product.  The edges of the stain are clearly outlined and the zone of fluorescence (the stain) is clearly distinct from the dark background (cloth, paper, etc.).The macroscopic aspect of this fluorescence allows one to believe—if not affirming the existence of semen—at least the probability of its existence through the analysis of its characteristics (the cut and contours of the stain, its limited imbibition by the cloth supporting it, the color of the fluorescence) compared with the fluorescence of other products, such as urine, saliva, sputum, etc.b)  The spectrographic analysis of the light of the fluorescence emitted by the stained cloth permits one to make a diagnosis of the presence or absence of semen.Fery’s spectrograph will show, with the direct transmission of Wood’s light through the stain, that semen has a fluorescent band spectrum ranging from 4.200 to 4.900 Ao.  This spectrum remains constant, even with different samples and stains of different ages.c)  The fluorescent spectrum of semen appears to us to be characteristic; it can be differentiated from that of other organic and inorganic products, such as urine, sputum, etc.The spectrography of semen stains leads to certainty for the following reasons:

    a) The fluorescent spectrum is characteristic

    b) The age of the semen stain does not diminish the fluorescence.

    c) The existence of azoospermia does not appear to modify the fluorescence of the semen.

  6. Biological Methods: As in the case of blood stains, one can use the methods of precipitating serums and anaphylaxis in order to determine if the semen is of human origin.  One can then proceed to the analysis of individual origin using Dervieux’s method.
  7. Vaseline: Semen stains are often mixed with Vaseline.  In that case, in the field of the microscope, one observes the Brownian movements of the fine birefringent particles.  A drop of potassium will not modify them, as would happen in the case of oily particles, but a drop of ether dissolves them.


Research investigating an abortion, infanticide or the suppression of a birth may lead to the discovery of soiled linens (sheets, towels, body wear, etc.), as well as mattresses or covers, which often have stains that are quite extensive.  Research will have to be carried out if one finds traces of blood, amniotic fluid, sebaceous coatings or meconium.

  1. Amniotic Fluid:These will take the form of extensive stains, ranging from very light yellow to violet, with an accentuated, grayish borders, and which weigh down the sheets.  Under the microscope, one will encounter rare, tile-shaped or polyhedral epithelial cells, fatty granulations and fetal hairs, which are small, colorless and lacking pigment, with a medullary canal that is scarcely visible and of a diameter that scarcely exceed 30 microns.
  2. Sebaceous Coating:This will form stains that are quite large, white, with a greasy look, and with gaps corresponding to the folds of the body.  Under the microscope, one will find a large number of epithelial cells that are tile-shaped or polyhedral, from 20 to 50 microns in diameter, lacking a clear nucleus, often overlapping, and which swell in glycerin.  In addition, one will find numerous fetal hairs.
  3. Meconium:These stains are brilliant, quite extensive, brown or dark brown, or dark green if they are thin, with a greenish-yellow border when they are fresh.  When these stains are dried, they are dull, fissured and have yellowish, scaly borders.  These stains are soluble in water, which makes them swell up and turns them greenish-yellow with darker lumps on the surface.  They react with biliary pigments (Gmelin’s reaction: In a test tube, azotic acid, in contact with  meconium dissolved in water yield rings that are violet, yellow, blue or green). In a chloroform solution, meconium and concentrated sulfuric acid will give a cholesterol reaction, that is, a greenish-yellow coloration with the acid at the bottom of the test tube and a pinkish coloration with the chloroform at the surface.When examined under a microscope, a bit of meconium swelled in water and dissociated with a needle will present grayish granulations, characteristic yellow-brown or greenish corpuscles, overlapping or prismatic crystals and fetal hairs that are easy to recognize. One can also use the method of precipitating serums or anaphylaxis to determine the origin of the meconium.


Stains from fecal materials can be found at the scene of a crime, where the guilty party has defecated either intentionally or through need or for superstitious reasons.  The stains can be found on paper that the criminal has used to wipe himself.  They may also be mixed with obstetrical stains or among the multiple stains that finds on the sheets of victims of sex crimes.

These stains vary in shape and nuance, but they will only be on one side of cloth or paper.  Through microscopic observation one will discover all kinds of food traces, products of gastro-intestinal secretions and also microbes and parasites.

A complete examination of fecal material is very difficult.  On the other hand, a microscopic examination will present some elements that are almost never deceptive.  One will almost always find yellow granules that appear to be the final product of the breakdown of muscle fibers (Schmidt and Strassburger), and also whitish granulations that are bits of coagulated casein.  Elastic fibers that are “fat, split or bifurcated, rolled up or lined up in rolls” are frequent, as well as conjunctive fibers that are “colorless, taut, isolated or joined in heaps.”  Debris from vegetables is extremely polymorphic and is often altered very slightly (grains of starch, vessels rolled or in spirals, vegetable fibers, polyhedral, spherical or spindle-shaped cells, some aligned like fence posts, etc.).  All of these will be bathed in mucous, in a granulated mass whose striations become pronounced in acetic acid.

Among the crystals, it is common to find: Charcot-Leyden crystals that are octahedral, very small and transparent; cholesterol crystals, shaped like rhomboid plates of varying size, with one corner chopped off and with one overlapping the next (See also Dervieux and Leclercq, op. cit., p. 307 and Rene Galutier, Outline of Clinical Coprology).

The preparation is made with glycerin.  One will see a bottom layer of granulations characterized by Brownian motion, the histological elements and the crystals on top of it.


Microscopic examination does not yield any certain results.  Chemical examination, using Balthazard’s method (Richard’s reactive with phosphotungstate of soda) leads to confusion with saliva and feces.  The only reliable test is that of Policard, using dixanthylurea.

Policard’s Method:  
From the stained area, cut a square measuring 2 mm. on each side and place it on a lamp, where the square will be dissociated in its dry form, using needles.  On the dissociated fibers, place one or two drops of the following solution, prepared on the spot: Equal parts of xanthydrol in a 93% solution of alcohol, and crystallizable acetic acid.

In order to make the xanthydrol solution, one weighs out one gram of the substance and places it in 100 cc of 93% alcohol; part of the xanthydrol will remain undissolved.  Then filter it before use.

Cover the preparation with a slide and make a hermetic seal using paraffin, since desiccation would impede the reaction.  Let the reactive work for about an hour and examine it under weak magnification.  If there is urine, crystals of xanthylurea will form in groups radiating around and between the fibers.  The reaction is quantitative; the abundance of crystals will be proportionate to the amount of urine present.

Fosse has proposed an analogous, but perhaps more sensitive, test.  Dissolve the stain in distilled water and let it soak for 24 hours.  Heat it slightly.  Add two volumes of acetic acid.  Add ½ cc of xanthydrol in a 10% solution of pure methyl alcohol.  One will obtain white crystals of xanthylurea after about half an hour.  These can be gathered through centrifugation.


On clothes or linens, one might encounter mucus stains that will be important to recognize in order to distinguish them, in particular, from semen stains.  One might be dealing with saliva, sputum from certain forms of bronchial mucus, nasal mucus, vaginal or uterine secretions or secretions from the prostate or urethra. 

  1. Saliva: These stains are gray, white or yellowish and weigh the cloth down slightly; they have irregular contours.  Microscopic examination of a stain soaked in distilled water will show tile-shaped epithelia cells of mucus from the mouth or pharynx, cylindrical cells with vibratile hairs of the respiratory tract, numerous microbes, in particular spirilla from the mouth, and alimentary debris.On the other hand, soaking the stain in hydrochloric acid will yield, in the presence of ferric chloride, a bright, blood-red stain, characterizing the presence of sulfo-cyanate of potassium, a constant element in saliva.  One should not fail to check for the presence of saliva on rags or cotton used as gags found on the victims who are bound and gagged; this sort of scenario is often faked.
  2. Bronchial Secretions:  These stains are filmy white or grayish, with fringed contours.  Microscopic examination will reveal a striated substratum formed of mucus, epithelial cells from respiratory mucus, prismatic cells with vibratile hairs, tile-shaped cells from the mouth or pharynx, diverse microbes and Charcot-Leyden crystals.
  3. Nasal: Mucus stains are gray, but sometimes yellow or greenish, and black when mixed with atmospheric dust, and have a weakly adhering crust.  Microscopic examination will reveal a striated substratum formed of mucus, prismatic cells from the nasal cavities epithelial cells with vibratile hairs, numerous microbes and particles of carbon.
  4. Vaginal:  Secretions stains are fairly extensive, yellowish and have clearly defined, sinuous contours; they weigh down the cloth and have a whitish, yellow or green crust.  Microscopic examination will reveal a reticulum of striated mucus which is rendered more clear with acetic acid; polyhedral, vaginal cells, rounded or lozenge-shaped, sometimes overlapping; with globules of pus and leucocytes.
  5. Secretions from the Prostate or Urethra: These stains are rounded, grayish or yellowish, not very extensive, and weigh down the cloth.  Microscopic examination reveals a finely striated, mucous reticulum, a small number of leucocytes, rare epithelial urethral cells.  These stains give the Barberio and Florence reactions, as with semen.

Editor’s Note

Locard follows on with Part VI: Other Stains, Microchemistry, and Starches.


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