FEDERAL SCHOOL OF MEDICAL LABORATORY

TECHNOLOGY (SCIENCE), JOS

SUPRAVITAL STAINS AND THEIR APPLICATION IN HEMATOLOGY

A SEMINAR

BY

DADEP RINRET DAVID

MLT/038/2021
 TITLE PAGE

A final year seminar on the topic SUPRAVITAL STAINS AND THEIR

APPLICATION IN HEMATOLOGY by DADEP RINRET DAVID with
registration number MLT/038/2021

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 TABLE OF CONTENT
TITLE PAGE............................................................................................................ii
TABLE OF CONTENT...........................................................................................iii
ABSTRACT.............................................................................................................iv
CHAPTER ONE........................................................................................................1
INTRODUCTION.....................................................................................................1
CHAPTER TWO.......................................................................................................4
LITERATURE REVIEW..........................................................................................4
2.1 New methylene blue.........................................................................................5
2.2 Crystal violet....................................................................................................6
2.2.1 Applications...............................................................................................7
2.2.2 Medical.......................................................................................................8
2.2.3 Veterinary...................................................................................................9
2.2.4 Synthesis....................................................................................................9
2.3 Methyl Voilet...................................................................................................9
2.3.1 History........................................................................................................9
2.4 Gentian violet.................................................................................................10
2.5 Advantages.................................................................................................

2.6 Disadvantages............................................................................................

2.7 Precautions.....................................................................................................11
CONCLUSION.......................................................................................................13
REFERENCES........................................................................................................14

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 ABSTRACT
In supravital staining the living cells take up the stain, although in "vital staining"
the living cells exclude the stain i.e. stain negatively and only the dead cells stain
positively and thus viability can be assessed by counting the percentage of total
cells that stain negatively. Staining is a technique used to enhance contrast in
samples, generally at the microscopic level. A combination of supravital and vital
dyes can also be used in a sophisticated way to better classify cells into distinct
subsets (e.g. viable, dead, dying etc.). The most common supravital stain is
performed on reticulocytes using new methylene blue or brilliant cresyl blue,
which makes it possible to see the reticulofilamentous pattern of ribosomes
characteristically precipitated in these live immature red blood cells by the
supravital stains. By counting the number of such cells the rate of red blood cell
formation can be determined, providing an insight into bone marrow activity and
anemia

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 CHAPTER ONE

INTRODUCTION

Hematology is the study of blood and blood disorders. Hematologists and

hematopathologists are specialists highly trained in the field as healthcare service
providers specialized in the components, compositions and diseases of the blood.
Some of these include blood and bone marrow cells. The hematological tests can
help diagnose anemia, infection, hemophilia, blood-clotting disorders, and
leukemia.

Vital stain

A vital stain in a casual usage may mean a stain that can be applied on living cells
without killing them. Vital stains have been useful for diagnostic and surgical
techniques in a variety of medical specialties. In supravital staining, living cells
have been removed from an organism, whereas intravital staining is done by
injecting or otherwise introducing the stain into the body. The term vital stain is
used by some authors to refer to an intravital stain, and by others interchangeably
with a supravital stain, the core concept being that the cell being examined is still
alive. In a more strict sense, the term vital staining has a meaning contrasting with
supravital staining. While in supravital staining the living cells take up the stain, in
"vital staining" – the most accepted but apparently paradoxical meaning of this
term, the living cells exclude the stain i.e. stain negatively and only the dead cells
stain positively and thus viability can be assessed by counting the percentage of
total cells that stain negatively. Very bulky or highly charged stains that don't cross
live plasma membrane are used as vital stains and supravital stains are those that
are either small or are pumped actively into live cells. Since supravital and

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intravital nature of the staining depends on the dye, a combination of supravital
and vital dyes can also be used in a sophisticated way to better classify cells into
distinct subsets (e.g. viable, dead, dying etc.). Eosin dye exclusion List of common
vital stains includes:

Propidium iodide, DNA stain that can differentiate necrotic, apoptotic and normal
cells. Trypan Blue, a living-cell exclusion dye Triphenyl tetrazolium chloride
Erythrosine, which is Red No. 3 in food coloring, can be used as an exclusion dye.
7-Aminoactinomycin D used e.g. in flowcytometric studies of hematopoietic stem
cell viability. Other Vital Stains are;

Janus Green B is a basic dye and vital stain used in histology.

Staining

Staining is a technique used to enhance contrast in samples, generally at the

microscopic level. Stains and dyes are frequently used in histology (microscopic
study of biological tissues), in cytology (microscopic study of cells), and in the
medical fields of histopathology, hematology, and cytopathology that focus on the
study and diagnoses of diseases at the microscopic level. Stains may be used to
define biological tissues (highlighting, for example, muscle fibers or connective
tissue), cell populations (classifying different blood cells), or organelles within
individual cells. A stained histological specimen, sandwiched between a glass
microscope slide. In biochemistry, it involves adding a classspecific (DNA,
proteins, lipids, carbohydrates) dye to a substrate to qualify or quantify the
presence of a specific compound. Staining and fluorescent tagging can serve
similar purposes. Biological staining is also used to mark cells in flow cytometry,
and to flag proteins or nucleic acids in gel electrophoresis. Light microscopes are
used for viewing stained samples at high magnification, typically using bright-field

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or epi-fluorescence illumination. Staining is not limited to only biological
materials, since it can also be used to study the structure of other materials; for

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example, the lamellar structures of semicrystalline polymers or the domain
structures of block copolymers.

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 CHAPTER TWO
LITERATURE REVIEW

Supravital staining is a method of staining used in microscopy to examine living

cells that have been removed from an organism. It differs from intravital staining,
which is done by injecting or otherwise introducing the stain into the body. Thus a
supravital stain may have a greater toxicity, as only a few cells need to survive
it a short while. The term "vital stain" is used by some authors to refer specifically
to an intravital stain, and by others interchangeably with a supravital stain, the
core concept being that the cell being examined is still alive. As the cells are
alive and unfixed, outside the body, supravital stains are temporary in
nature. Supravital stain of a smear of human blood from a patient with hemolytic
anemia. The reticulocytes are the cells with the dark blue dots and curved linear
structures (reticulum) in the cytoplasm. The most common supravital stain is
performed on reticulocytes using new methylene blue or brilliant cresyl blue,
which makes it possible to see the reticulofilamentous pattern of ribosomes
characteristically precipitated in these live immature red blood cells by the
supravital stains. By counting the number of such cells the rate of red blood cell
formation can be determined, providing an insight into bone marrow activity and
anemia. This is in contrast to vital staining, when the dye employed is one that is
excluded from the living cells so that only dead cells are stained positively. (Vital
stains include dyes like trypan blue and propidium iodide, which are either too
bulky or too charged to cross the cell membrane, or which are actively rapidly
pumped out by live cells.) Supravital staining can be combined with cell surface
antibody staining (immunofluorescence) for applications such as FACS analysis.
Immunofluorescence can also be done within the interior of live cells by reversible
cell permeabilization using the detergent Triton X-100. Adjusted carefully to the

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appropriate concentration for the number of cells, the pretreatment can permit
access of molecules between 1 and 150 kilodaltons to the interior of the cell.
Although antibodies may be used in a similar way in this context, the term
"supravital stain" is typically reserved for smaller chemicals which possess suitable
properties intrinsically.
Supravital dyes for RBCs:
i. New methylene blue
ii. Brilliant cresyl blue
iii. Crystal violet
iv. Methyl violet
v. Nile blue
vi. Hoechst stain

2.1 New methylene blue

New methylene blue (also NMB) is an organic compound of the thiazine class of
heterocycles. It is used as a stain and as an antimicrobial agent. It is classified as an
azine dye, and the chromophore is a cation, the anion is often unspecified.
NMB is a staining agent used in diagnostic cytopathology and histopathology,
typically for staining immature red blood Boiling cells. It is a supravital stain. It is
closely related to methylene blue, an older stain in wide use.
New methylene blue is toxic. Skin contact or inhalation should be avoided.

2.2 Crystal violet

Crystal violet or gentian violet, also known as methyl violet 10B or hexamethyl
pararosaniline chloride, is a triarylmethane dye used as a histological stain and in
Gram's method of classifying bacteria. Crystal violet has antibacterial, antifungal,
and anthelmintic (vermicide) properties and was formerly important as a topical

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antiseptic. The medical use of the dye has been largely superseded by more
modern drugs, although it is still listed by the World Health Organization.
Crystal violet

The gentian violet was originally used for a mixture of methyl

pararosaniline dyes (methyl violet), but is now often considered a synonym for
crystal violet. The name refers to its colour being like that of the petals of certain
gentian flowers; it is not made from gentians or violets. A number of possible
routes can be used to prepare crystal violet. The original procedure developed by
the German chemists Kern and Caro involved the
reaction of dimethylaniline with phosgene to give 4,4′bis(dimethylamino)
benzophenone (Michler's ketone) as an intermediate.
This was then reacted with additional dimethylaniline in the presence of
phosphorus oxychloride and hydrochloric acid.
Crystal violet (pH indicator) below pH −1.0 above pH 2.0 −1.0 ⇌ 2.0
When dissolved in water, the dye has a blue-violet colour with an absorbance
maximum at 590 nm and an extinction Solid crystal violet Crystal . The colour of
the dye depends on the acidity of the solution. At a pH of +1.0, the dye is green
with absorption maxima at 420 nm and 620 nm, while in a strongly acidic solution
(pH −1.0), the dye is yellow with an absorption maximum at 420 nm. The different
colours are a result of the different charged states of the dye molecule. In the
yellow form, all three nitrogen atoms carry a positive charge, of which two are
protonated, while the green colour corresponds to a form of the dye with two of the
nitrogen atoms positively charged. At neutral pH, both extra protons are lost to the
solution, leaving only one of the nitrogen atoms positive charged. The pKa for the
loss of the two protons are approximately 1.15 and 1.8. In alkaline solutions,

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nucleophilic hydroxyl ions attack the electrophilic central carbon to produce the
colourless
triphenylmethanol or carbinol form of the dye. Some triphenylmethanol is also
formed under very acidic conditions when the positive charges on the nitrogen
atoms lead to an enhancement of the electrophilic character of the central carbon,
which allows the nucleophilic attack by water molecules. This effect produces a
slight fading of the yellow colour.

2.2.1 Applications
Supravital staining is a good technic for the identification of monocytes from
human blood and marrow, and is the method of choice for the study of
macrophages.The dye is used as a histological stain, particularly in Gram staining
for classifying
bacteria. When conducting DNA gel electrophoresis, crystal violet a nontoxic
DNA stain as an alternative to fluorescent, intercalating dyes such as ethidium
bromide. Used in this manner, it may be either incorporated into the agarose gel or
applied after the electrophoresis process is finished. Used at a 0.001%
concentration and allowed to stain a gel after electrophoresis for 30 minutes, it can
detect as little as 16 ng of DNA. Through use of a methyl orange counterstain and
a more complex staining method, sensitivity can be improved further to 8 ng of
DNA. When crystal violet is used as an alternative to fluorescent stains, it is not
necessary to use ultraviolet illumination; this has made crystal violet popular as a
means of avoiding UV-induced DNA destruction when performing DNA cloning
in vitro. In biomedical research, crystal violet can
be used to stain the nuclei of adherent cells. In this application, crystal violet
works as an intercalating dye and allows the quantification of DNA which is

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proportional to the number of cells. In forensics, crystal violet was used to develop
fingerprints. Crystal violet is also used as a tissue stain in the preparation of light
microscopy sections. In laboratory, solutions containing crystal violet and formalin
are often used to simultaneously fix and stain cells grown in tissue culture to
preserve them and make them easily visible, since most cells are colourless. It is
also sometimes used as a cheap way to put identification markings on laboratory
mice; since many strains of lab mice are albino, the purple colour stays on their fur
for several weeks. In body piercing, gentian violet is commonly used to mark the
location for placing piercings, including surface piercings. Marking blue, used to
mark out pieces in metal working, is composed of methylated spirits, shellac, and
gentian violet.

2.2.2 Medical
Gentian violet has antibacterial, antifungal, antihelminthic, antitrypanosomal,
antiangiogenic, and antitumor properties. It is used medically for these properties,
in particular for dentistry, and is also known as "pyoctanin" (or "pyoctanine"). It is
commonly used for: Marking the skin for surgery preparation and allergy testing;
Treating Candida albicans and related fungal infections, such as thrush, yeast
infections, various types of tinea (ringworm, athlete's foot, jock itch); Treating
impetigo; it was used primarily before the advent of antibiotics, but still useful to
persons who may be allergic to penicillin. In resource-limited settings, gentian
violetis used to manage burn wounds, inflammation of the umbilical cord stump
(omphalitis) in the neonatal period, oral candidiasis in HIV-infected patients and
mouth ulcers in children with measles.

2.2.3 Veterinary
Because of its antimicrobial activity, it is used to treat fish. However, it usually is
illegal to use in fish intended for human consumption.

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2.2.4 Synthesis
Crystal violet is one of the components of methyl violet, a dye first synthesized by
Charles Lauth in 1861. From 1866,

2.3 Methyl Violet

2.3.1 History
methyl violet was manufactured by the Saint-Denis-based
firm of Poirrier et Chappat and marketed under the name "Violet de Paris".
It was a mixture of the tetra-, penta- and hexamethylated pararosanilines. Crystal
violet itself was first synthesized in 1883 by Alfred Kern (1850–1893) working
in Basel at the firm of Violetchedler and Busch. To optimize the difficult synthesis
which used the highly toxic phosgene, Kern entered into a collaboration with the
German chemist Heinrich Caro at BASF. Kern also found that by starting with
diethylaniline rather than dimethylaniline, he could synthesize the closely related
violet dye now known as C.I. 42600 or C.I. Basic violet 4.

2.4 Gentian violet

The name "gentian violet" (or Gentianaviolett in German) is thought to have been
introduced by the German pharmacist Georg Grübler, who in 1880 started a
company in Leipzig that specialized in the sale of staining reagents for histology.
The gentian violet stain marketed by Grübler probably contained a mixture of
methylated pararosaniline dyes. The stain proved popular and in 1884 was used by
Hans Christian Gram to stain bacteria. He credited Paul Ehrlich for the aniline-
gentian violet mixture.Grübler's gentian violet was probably very similar, if not
identical, to Lauth's methyl violet, which had been used as a stain by Victor André
Cornil in 1875. Although the name gentian violet continued to be used for the
histological stain, the name was not used in the dye and textile industries. The
composition of the stain was not defined and different suppliers used different

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mixtures. In 1922, the Biological Stain Commission appointed a committee chaired
by Harold Conn to look into the suitability of the different commercial products. In
his book Biological Stains, Conn describes gentian violet as a "poorly defined
mixture of violet rosanilins". The German ophthalmologist Jakob Stilling is
credited with discovering the antiseptic properties of gentian violet. He published a
monograph in 1890 on the bactericidal effects of a solution that he
christened "pyoktanin", which was probably a mixture of aniline dyes similar
to gentian violet. He set up a collaboration with E. Merck & Co. to market
"Pyoktanin caeruleum" as an antiseptic. In 1902, Drigalski and Conradi found that
although crystal violet inhibited the growth of many bacteria, it has little effect on
Bacillus coli (Escherichia coli) and Bacillus typhi (Salmonella typhi), which are
both gram-negative bacteria. A much more detailed study of the effects of
Grübler's gentian violet on different strains of bacteria was published by John
Churchman in 1912. He found that most gram-positive bacteria (tainted) were
sensitive to the dye, while most gram negative bacteria (not tainted) were not,
and observed that the dye tended to act as a bacteriostatic agent rather than a
bactericide.

2.5 Advantages
In enumerating the advantages of the supravital staining, then, we find that it
demonstrates the mitochondria, granules, vacuoles or crystals usually
unrecognizable in fixed preparations; it permits of the direct observation of such
physiological activities of the cells as locomotion and phagocytosis.
It also provides more cytological details of the cell, where specific organelles can
be stained.

2.6 Disadvantages
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Most of these stains are dyes and are extremely toxic, which can result in death, so
it has to be diluted to a larger extent.

2.7 Precautions
One study in mice demonstrated doserelated carcinogenic potential at several
different organ sites.The Food and Drug Administration in the US (FDA) has
determined that gentian violet has not been shown by adequate scientific data to be
safe for use in animal feed. Use of gentian violet in animal feed causes the
feed to be adulterated and is a violation of the Federal Food, Drug, and Cosmetic
Act in the US. On June 28, 2007, the FDA issued an "import alert" on farm raised
seafood from China because unapproved antimicrobials, including gentian violet,
had been consistently found in the products. The FDA report states: "Like MG
(malachite green), CV (crystal violet) is readily absorbed into fish tissue
from water exposure and is reduced metabolically by fish to the leuco moiety,
leucocrystal violet (LCV). Several studies by the National Toxicology Program
reported the carcinogenic and mutagenic effects of crystal violet in rodents. The
leuco form induces renal, hepatic and lung tumor in mice. "Health Canada recently
found medical devices that use gentian violet to be safe for use but recommended
to stop using all drug products that contain gentian violet, including on animals,
causing Canadian engineering schools to revisit the usage of this dye during
orientation.

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 CONCLUSION
When there is an increased production of red blood cells to overcome chronic or
severe loss of mature red blood cells, people often have a markedly high number
and percentage of reticulocytes. A very high number of reticulocytes in the blood
can be described as recticulocytosis.
Supravital staining of suspicious mucosal lesions in oral cavity helps in the
early diagnosis of malignant lesions. These malignant lesions of mucosa have large
amounts of acidic DNA and RNA due to the active cellular proliferation which is
occurring there. This dye is acidophilic in nature and preferentially stains acidic
cellular components like DNA and RNA. Supravital staining can be combined with
a cell surface antibody staining for applications such as Facs Analysis. Simple
supravital staining technique can be used with ordinary BFM for accurate urine
sediment analysis in cases of ARF in bedside medicine.

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 REFERENCES

Harris J.E. and Peters A. (1953). Experiments on vital staining with methyl blue,
Q. J. Microsc Sci. 94, 113-124.
Kiernan J.A. (1974). Effects of metabolic inhibitors on vital staining with
methylene blue. Histochemistry 40, 51-57
Simpson M.E. (1921). Vital staining of human blood with special reference to the
separation of the monocytes. University of Califonia publications in
Anatomy
Casey A. E. and Roshan, P. D. (1981). Delayed differential counting of white
blood cells by a modified supravital technique.
John P. Greener; Maxwell Myer Wintrobe (2008). “Wintrobe’s clinical
hematology
Kiernan J.A. (2001). Classification and naming of dyes, stains and flourochromes”.
Biotechnic and Histochemistry.
Wells J. (1988). “ A Technique for staining the superficial cells of plucked hair
follicles and other solid tissues”. Stain technology
Bancroft J. Stevens (1982). The theory and practice of Histological Techniques (2 nd
ed.). Longman group limited.

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