Introduction:

Fecal matter is not only examined microscopically but as well as macroscopically and chemically
because these examinations can also be of help in diagnosing a disease.

Objectives:
After performing this exercise, the student should be able to:
1. macroscopically describe the appearance of a normal and abnormal stool; and
2. do or perform and interpret chemical tests for stool samples.

Materials:
8% H2O2 Stool specimen
Nitrazine Paper
Benzidine solution
Hema - screen kit
10% Barium chloride aqueous solution

Procedure:

I. Macroscopic Examination

1. Prepare the start of tests, clean the working area and cover it with newspaper enough to
hold your specimen.
2. Open the container of stool sample aseptically to avoid contaminating hands. Place cover
of container on the covered area side up.
3. Observe color, odor, form and consistency of stool sample (stab the sample with an
applicator stick).
4. Note the presence of adult parasite or proglotidds, undigested food, fibers, concretions,
mucus, blood streak and fats.
5. Specimen may be liquefied with distilled water and strained through a clean wire mesh
sieve or nylon sieve (wire or nylon screen) to recover smaller worms and scolices.

II. Chemical Examinations

A. pH (Nitrazine Paper test)
Procedure:
1. With an applicator stick, smear a small amount of fecal emulsion on piece of nitrazine
paper.
2. Wait for two minutes then observe color change and compare with color chart.
 B. Occult Blood
a) Benzidine Test
Principle:
The peroxidases contained in the heme portion of the blood react with H 2O2 liberating
oxygen which in turn oxidizes benzidine to form a blue colored compound.

Procedure:
1. With an applicator stick, smear a small amount of stool on a clean filter paper.
2. Add 2 drops of 8% H2O2 on the smear and then add also two drops of benzidine solution.
3. Let stand for about 30-60 seconds and note the color change.

Interpretation:

No color change - Negative

Greenish linge -Trace

Light green -1+

Dark green -2+

Blue green -3+

Deep blue -4+

b) Hema-screen

Principle:
When stool specimens containing occult blood are applied to hema-screen test paper, the
hemoglobin portion of the occult blood comes in contact with the guaiac. When the hema-
screen peroxide developing solution is added, a guaiac-peroxidase like reaction occurs. The
chemical reaction becomes visible by the appearance of a blue-green color between 30 seconds
if occult blood is present.

Procedure:
1. With an applicator, apply very thin smear of stool the area indicated with roman numeral I.
2. Using the same applicator repeat from a different portion of the stool for area II. Discard the
applicator in a container with lysol or hypochlorite.
3. On back of slide, open perforated section, marked 1 and 2.
4. Apply two or more drops of hema-screen developing solution to exposed test paper.
5. Read results between 30-60 seconds.

Interpretation:
 Any trace of Blue – Positive for occult blood
 No indication of blue – Negative
c) Test for Pigments (Gmelin’s Test)

Principle:
After precipitation of bile pigments by barium chloride, it is oxidized by nitric acid to derivatives
such as beliverdin (green), bilicyanin (blue) and choletelin (yellow).

Procedure:
1. Place 5 ml. of fecal emulsion in a test tube.
2. Add 5 ml. 10% aqueous solution of barium chloride. Mix, let stand for a few minutes and
filter.
3. Allow the filter paper to dry partially then add yellow nitric acid.

Interpretation:
A positive reaction is indicated by a play of colors, green on periphery, then in order toward
center, blue, violet, red and yellow. The absence of green excludes the presence of bile pigments.

Guide Questions:

1. What is/are responsible for the offensive odor of stool?

The presence of certain compounds produced by gut bacteria during the digestion of food are
responsible for the offensive odor of the stool as analyzed by Sato et al. (2002). These compounds
include skatole, indole, putrescine, thiols, and mercaptans. First, skatole is a white, crystalline
compound produced by gut bacteria during the breakdown of amino acids. Another would be the indole
which is a colorless, crystalline compound with a strong, unpleasant odor that is produced by gut
bacteria during the breakdown of tryptophan. Furthermore, putrescine is also from the breakdown of
amino acids. Thiols are a class of organic compounds that contain a sulfur group and are responsible for
the rotten egg smell of some stools. Mercaptans are sulfur-containing organic compounds that are also
produced by gut bacteria during the breakdown of sulfur-containing amino acids and have a strong,
sulfurous odor. Additionally, the odor of stool can be attributed to various factors such as diet,
medication, and health conditions. However, a foul-smelling stool—having a putrid and rotten odor—
can be caused by certain foods, medications, or a concerning indication of underlying gastrointestinal
disorders such as infections, inflammatory bowel disease (IBD), and malabsorption; often accompanied
by diarrhea, bloating, and flatulence, this should be further evaluated by a healthcare professional
(Kahn, 2020).

2. What is the normal color of babies’ stool? Why?

Dr. Sherman (2020) claims that brown, yellow, and green are normal and may often variate
between those. The normal color of babies' stool can vary depending on their diet. If a baby is breastfed,
their stool may be yellow or green and have a seedy, runny consistency. This is known as breastmilk
stool and is considered normal. If a baby is formula-fed, their stool may be darker or tan and have a
thicker, pasty consistency. The reason for these color variations is due to the different pigments and
nutrients found in breast milk and formula. Additionally, meconium, a dark greenish-black, tar-like
substance, is the first stool passed by newborns, which contains amniotic fluid, bile, and lanugo among
other substances that the baby ingested while in the uterus. Red stool from blood streaks, chalk white
stool from the lack of bile, and black stool of children more than three years old warrant a clinical
consultation.
 I. Observation/Results

Figure 1. The stool's color appears to be greenish

brown in natural light. When poked with an
applicator stick, it has a diarrheal consistency.

Figure 2. The MT students inoculated the

sample onto the Fecal Occult Blood tester using
a sample extractor with an integrated dropper.

Figure 3. After the students dropped a

significant amount of sample from the sample
extractor, they waited a short while until the
FOB tester revealed two lines, indicating that
the feces was positive for blood.
II. Discussion
Differentiating normal and abnormal stools through macroscopic examination can provide
important insights into determining the presence of pathological conditions in the gastrointestinal tract
(Kasırga, 2019) (Kano, 2021). A normal stool is typically a consistent brown color, which is due to the
presence of bilirubin. However, abnormal stools can vary significantly in color, form, and consistency.
For example, a dark green stool may indicate rapid transit through the intestines or celiac disease. In
contrast, a black or tarry stool can indicate bleeding from the upper gastrointestinal tract while a bright
red stool can indicate bleeding from the lower gastrointestinal tract. Additionally, a white or clay-
colored stool could indicate a lack of bile in the stool or a liver disorder. Furthermore, the appearance of
the stools’ form and consistency can also be important in diagnosing gastrointestinal disorders. A well-
formed, soft stool is typically considered normal. In contrast, a hard, lumpy stool may be an indication of
constipation or a lack of fiber in the diet while consistent watery stools that can pour is a sign of
diarrhea. Other abnormalities that may be observed macroscopically include the presence of undigested
food particles, excess mucus, blood, worms, scolices of tapeworms, or fat. These findings may indicate
the presence of an infection or disease, such as ascariasis or hemorrhoids.
Chemical examinations such as pH testing, occult blood testing, and pigment analysis offer a
vital tool for the diagnosis of gastrointestinal disorders. First, is the pH testing or the pH fecal test, in
which, a fecal specimen is collected and tested for acidity to diagnose a medical condition (Nichols et al.,
2006). The normal range for stool pH is slightly acidic, slightly alkaline, or neutral, which is between 5.8
to 7.5. If the stool pH is below 5.5, it is strongly acidic, which could indicate too much carbohydrates in
the diet or digestive issues like lactose intolerance; or a contagion such as E. coli or rotavirus.
Conversely, if the stool pH is above 7.5, it is strongly alkaline, which could indicate too much protein in
the diet. To perform the test, a strip of nitrazine paper is dipped in at least half a milliliter of fecal
sample; after some time, a resulting color change is observed and compared to a chart. However, it is
important to note that the patient must not be receiving antibiotics, as this can affect the results of the
test.
Second, there is the occult blood test which is formulated to determine blood in sample that are
not readily visible to the eye (Kaur & Adamski, 2021). One such test is the benzidine test, also known as
Wagner test, Jada et al. (2015) explains that it utilizes the peroxidases contained in the heme portion of
blood to react with hydrogen peroxide, liberating oxygen which in turn reacts to benzidine to form a
blue colored compound. To create a benzidine solution, combine 0.25 g of benzidine with 2.5 ml of
glacial acetic acid and water in an equal amount, and 2 ml of 3% hydrogen peroxide. A few drops of the
benzidine solution can be added after a thin layer of the fecal sample is spread out on a glass. A deep
green or blue color that appears within a minute indicates that the test was reactive or positive. Another
occult blood test is the Hema-Screen test. Wakid (2010) considers it a guaiac-based test and it is
intended for use in professional settings to diagnose asymptomatic gastrointestinal conditions.
Specifically, the test uses hemoglobin's peroxidase-like activity to cause a chemical reaction visible as a
blue-green color in 30 to 60 seconds. False positives can result from improper diet, blood on hands,
hemorrhoids, or menstrual bleeding. The test helps detect colorectal cancer, ulcers, polyps, anemia, and
diverticulosis, and eliminates mess and odors associated with fecal specimens.
Lastly, the test for pigments, or Gmelin’s Test, is used to detect bile pigments in stool samples,
which can indicate gastrointestinal disorders such as liver disease or bile duct obstruction (Kuntz &
Kuntz, 2009). The test works by precipitating bile pigments with barium chloride and then oxidizing
them with nitric acid to produce derivatives that have distinct colors. To perform the test, a fecal
emulsion is mixed with barium chloride solution, filtered, and treated with yellow nitric acid. A positive
reaction is indicated by a play of colors including green, blue, violet, red, and yellow with the absence of
green ruling out the presence of bile pigments.

III. Conclusion

In conclusion, the examination of stool samples plays a crucial role in the diagnosis and
treatment of gastrointestinal disorders. Macroscopic examination, which includes observation of the
color, odor, form and consistency of the stool, can provide valuable information about the health of the
patient. A normal stool is typically brown in color, has a characteristic odor, and is well-formed and soft
in consistency. However, abnormal stools can present with a wide range of colors, odors, forms, and
consistencies, which can indicate various pathological conditions. Chemical testing of stool samples,
such as pH testing, occult blood testing, and pigment analysis, is also an important diagnostic tool for
gastrointestinal disorders. For instance, a pH test can indicate the presence of acid or alkaline stool,
which can be an indication of certain conditions. Similarly, occult blood testing can detect the presence
of blood in the stool, which can be an indication of a gastrointestinal bleed. Bile pigment analysis, such
as Gmelin's test, can detect the presence of abnormal pigments in the stool, which can be an indication
of certain conditions, such as biliary obstruction or liver disease. The offensive odor of stool can be
caused by the presence of volatile sulfur compounds, produced by the breakdown of proteins in the gut
by bacteria, and other factors such as poor digestion, certain medical conditions, and dietary factors.
The normal color of babies' stool can vary depending on their diet, with breastfed babies having a yellow
or green, seedy, runny consistency and formula-fed babies having a tan or yellow-brown, thicker, pasty
consistency. The presence of blood, pus, mucus, and fats in stool can indicate pathological conditions
and diseases such as gastrointestinal bleeding for blood, infection for pus, inflammation for mucus, or
fat malabsorption for fats. Overall, the importance of studying the macroscopic and chemical
examination of stool in medical/clinical practice cannot be overstated as it plays a critical role in the
diagnosis and treatment of various gastrointestinal diseases.

IV. References

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malodorous substances of human feces. Journal of health science, 48(2), 179-185.

Kahn, A. (2020, July 22). What causes foul-smelling stools? Healthline. Retrieved February 12, 2023, from
https://www.healthline.com/health/stools-foul-smelling

Sherman, J. (2020, December 31). The color of baby poop and what it means. Cleveland Clinic. Retrieved
February 13, 2023, from https://health.clevelandclinic.org/the-color-of-baby-poop-and-what-it-means-
infographic/

Boyle, J. T. (2008). Gastrointestinal bleeding in infants and children. Pediatrics in review, 29(2), 39.

Glynn, M., & Drake, W. M. (2017). Hutchison's Clinical Methods E-Book: An Integrated Approach to
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Stern, R. C., Eisenberg, J. D., Wagener, J. S., Ahrens, R., Rock, M., & Orenstein, D. M. (2000). A
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Kaur, K., & Adamski, J. J. (2021). Fecal Occult Blood Test. In StatPearls [Internet]. StatPearls Publishing.

Jada, S., Jayakumar, K., Vinoth, R., Ravichandran, R., Sivanandham, N., & Seeda, C. (2015). Detection of
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Wakid, M. H. (2010). Fecal occult blood test and gastrointestinal parasitic infection. Journal of
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Kuntz, E., & Kuntz, H. D. (2009). Hepatology: Textbook and atlas. Springer Science & Business Media.

Nichols, J. H., Taylor, D., Varnholt, H., & Williams, L. (2006). pH testing. Washington, DC: National
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Kasırga, E. (2019). The importance of stool tests in diagnosis and follow-up of gastrointestinal disorders
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Kano, Y. (2021). Campylobacter enterocolitis: the importance of macroscopic and microscopic

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