Microbiology- The

Latest on Staining
A Presentation By
Caitlyn Davis and
Shyamalee Ramaraj

Introduction
With the development of the microscope came the need to
distinguish microscopic organisms from their environment. By
themselves, they were nearly invisible, containing transparent
cytoplasm(s), therefore they were unable to be seen even with an
aided eye. Staining resolved this problem by enhancing the
visualization of cells by increasing contrast of the images of specimen
seen under the microscope, as well as pave way for the study of
microbe morphology, arrangement, structure, size, metabolism, and
behavior. Through a variety of staining methods, various strains of
microbes have been identified and utilized in the research fields of
aquatic, food, and medical microbiology. Many microbiological
advancements today can be attributed to proper applications of
staining.

Introduction (cont.)
This presentation aims to outline the significance of the
application or procedures of staining methods and
showcase past and present staining techniques in
microbiology.

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Objectives
The objectives of this presentation include the following:
To review basic staining procedures discussed in the laboratory
To introduce new methods employed today in individual cells of
tissues, viruses, and isolated culture samples of eukaryotes and
bacteria
To understand the purposes and importance of simple, differential,
and specialized staining and its implications in areas such as
aquatic, medical, and food microbiology as well as epidemiology
and etiology
To help one to comprehend the value of staining and instill
interest and appreciation for the field of microbiology and its
developments.

Outline/Overview of Presentation
This presentation will be delivered in the following manner beyond
this point:
1.

Video Virus staining

2.

Quick content review of staining methods

1.

Purpose of Staining

2.

Staining methods summary table

3.

Knowledge inventory

4.

Other stains (not discussed in laboratory)

5.

Video Fluorescence staining

3.

Evolution of Staining procedures in Microbiology + Names to

Remember

4.

Latest innovations in staining (main focus)

5.

Take home lesson(s)

 Short Video- Negative stains and Viruses

Staining, as mentioned previously, is important in the

identification of bacteria cells, eukaryotic cells, and their
constituent components within them (i.e., organelles). The
application of certain light and dye combinations can even
allow for the identification of viruses within host cells.
Morphology, size, and arrangement of targeted cells can
be determined through staining procedures.
Stains make specimens visible. Many sample cells on
smears are not visible to the naked eye and seem
especially transparent even under the microscope. The
addition of dyes allow for the coloring of inside the cells or
for the negatively-charged microbe to attract or repel the
stain, thereby filling the organism with color, or
surrounding it and increasing contrast.

 Such properties of stains in the laboratory also

make them useful and effective in fields of medical
and food microbiology for the classification,
separation, and elimination of good from bad
bacteria/organisms.
Various stains indicate the presence of different
organisms, thus certain types of stains target cells
that they are specific to. Knowing and
understanding the functions of specific stains can
allow one to wisely and correctly utilize them on a
particular type/aggregation of cells without
destroying its/their structure in order to study the
specimen.

Method of
Staining

Separates
specimen into

Live/dead
specimen?

Primary Stain
(negative or
basic)

Mordant
and/or
decolor-izer

Counterstain

Notes (simple,
special or
differential
staining):

Simple Staining

Organism vs. nonorganism (organism

vs. surroundings)

Dead specimen

Methylene blue,
safranin, or
crystal violet
(basic stains)

None.

None.

Simple staining,
basic stain
(positively
charged stain is
used), allows for
contrast between
microbe and
surrounding
space.

Gram-Staining

Gram-Positive vs.
Gram-Negative

Dead specimen

Crystal violet
(basic stain)

Gram iodine
(mordant),
acid alcohol
(decolorizer)

Safranin

Differential
staining, (+) has
thicker
pepdidoglycan
sublayer than (-).

Acidfast
Staining

Acidfast
(mycobacteria) vs.
non-acidfast

Dead specimen

Carbolfuchsin
(basic stain),
detergent
Tergitol is added
to primary stain
in heatless
method.

No mordant.
Acid alcohol
removes color
from nonacidfast
microbes.

Methylene blue or
brilliant green

Differential
staining,
separates bacteria
into those that
contain mycolic
acid in their outer
cell walls vs. ones
that do not.

Endospore
Staining

Endospore vs.
vegetative cells

Metabolically
inactive

Malachite green
(basic stain)

No mordant.
Water may
act as a
decolorizer.

Safranin

Differential
staining,
separates
organisms
containing
endospores from
vegetative cells.

Negative
Staining

Microbe (delicate)
vs. surroundings

Live specimin

Eosin or nigrosin
(negative or
acidic stains).

None.

None.

Special staining,

Flagellar
staining

Motile microbes vs.

surroundings

Live specimen

Carbolfuchsin or
pararosaliline
(basic stains).

Potassium
alum or
tannic acid.

None.

Special staining,

Quiz time!
1.

(1 skittle) What color is methylene blue?

2.

(2 skittles) Is carbolfuchsin a mordant? What is a

mordant?

3.

(3 skittles) What kind of stain (basic or acidic) is used

to identify viruses under a TEM in the previous video?

Some stains that we have not

yet discussed include:
1.

Bismark Brown (mucins)

2.

Carmine (glycogen)

3.

Coomassie blue (proteins)

4.

DAPI (fluorescent)

5.

Ethidium bromide (apoptosis)

6.

Hematoxylin (nucleus)

7.

Hoechst stains (fluorescent, DNA)

8.

Iodine (starch)

9.

India ink

10. Neutral Red (nucleus)

11. Nile red (lipids)
12. Nile blue (nucleus)
13. Osmium tetroxide (lipids)
14. Rhodamine (proteins)
15. Ammonium molybdate
16. Uranyl acetate
17. Uranyl formate
18. Phosphotungstic acid
19. Osmium ferricyanide
20. Auroglucothionate
21. Giemsa (pathogens, specifically viruses)
22. Acridine orange (DNA)
23. Cresyl violet (histology)
24. Methyl green (fluorescent, chromatin)
25. SYBR Green I Fluorescent stain(s)

Video - Fluorescence staining in medicine

Advancements in
Staining Procedures
At-A-Glance

16001750

Anton van
Leeuwenhoek uses
spice saffron to
stain muscle tissue

18301840

Discovery of
Fluorescein (1838,
1845)

18411860

Discovery,
synthesis, and
utilization of
carmine stain
(1854)

18601879

First Fluorescein
dye used in a
laboratory
setting (1871)

Staining with
metal
compounds
Silver staining
(1873)

18801890

Development of
Acid-fast
staining (1882,
publishing in
1883)

Gram staining
developed and
used (1883)

Ziehl-Neelsen
modification for
Acid-Fast
staining (1883)

18911910

Discovery of
hematoxylin and
eosin stains
(1896)

Giemsa stain
(1904)

Jensens
modification for
Gram staining
(1909)

19111925

Kinyons AcidFast method

modification
(1915)

Endospore
staining method
development
(1922)

Kopeloff and
Beermans Gram
Stain
modification
(1922)

19401960

Utilization of
Fluorochrome

Preston and
Morrells
modification of
Gram Staining
(1951)

First viewing of
virus after
applying stain
(1887)

Cytochemiical
staining of
peroxidase
(1924)

Weigerts
modification of
Gram Staining
(1887)

1800

1810

1820

1830

1840

Discovery of Fluorescein (1838, 1845)

Fluorescein is a fluorophore commonly used in microscopy, in a
type of dye laser as the gain medium, in forensics and serology
to detect latent blood stains, and in dye tracing. In cellular
biology, the isothiocyanate derivative of fluorescein is often
used to label and track cells in fluorescence microscopy
applications (for example, flow cytometry). Additional biologically
active molecules (such as antibodies) may also be attached to
fluorescein, allowing biologists to target the
fluorophore to
specific proteins or structures within cells.

1850

1860

1870

1880

1890

First Fluorescein dye used in a laboratory setting (1871)

With the development of the synthetic dye industry, it was not long before Adolf von Bayer
synthesized the first fluorescent dye, fluorescein.
Development of Acid-fast staining (1882, publishing in 1883)
Paul Erlich developed acid-fast staining in 1882 while working with Mycobacterium tuberculosis.
Ziehl and Neelson later improved the technique by adding heat to drive carbol fuchsin into the cell
wall.
Gram staining developed and used (1883)
In 1883-4, Dr. Gram discovered a method for staining certain bacterial cells purple/blue while
staining eukaryotic cells, and many other bacterial cells pink. Essentially, Dr. Gram found
that
bacteria fell into two distinct categories when stained
sequentially with crystal violet followed in
sequence by a bath in an iodine solution, a wash with a destaining agent and a
counter-stain.
First viewing of virus after applying stain (1887)
In 1887, Buist J.B. visualised one of the largest, Vaccinia virus, by optical microscopy after
staining it. Vaccinia was not known to be a virus at. that time

1900

1910

1920

1930

1940

Giemsa stain (1904)

Giemsa stain, named after German chemist and bacteriologist Gustav Giemsa,
is used in cytogenetics and for the histopathological diagnosis of malaria and
other
parasites. Giemsa stain is used in Giemsa banding, commonly called Gbanding,
to stain chromosomes and often used to create a karyogram
(chromosome map).

Endospore staining method development (1922)

In 1922, Dorner published a method for staining endospores. It employed a
lengthy heating step but resulted in differential staining of endospores and vegetative
cells in the same sample. Endospores and free spores appeared green
or blue in
contrast to red dye taken up by the vegetative cells.

Utilization of Fluorochrome (1940-1950)

A fluorophore (or fluorochrome, similarly to a chromophore) is
a fluorescent
chemical compound that can re-emit light upon
light excitation. Fluorophores
typically contain several combined aromatic groups, or plane or cyclic molecules
with several bonds. Fluorophores are sometimes used alone, as a tracer in fluids,
as a dye for
staining of certain structures, as a substrate of
enzymes, or as a
probe or indicator (when its fluorescence is affected by environmental aspects such
as polarity or ions).

Tried-and-tested staining techniques such as Gram and Acid-fast staining

have seen decades of use in the science of microbiology. At the same
time though, perpetual research in staining methods- both old and novelfor staining continue to be developed in pursuance of identifying,
isolating and studying new and old microbes alike; this includes
components relating to samples of multicellular organisms.
Some recent methods/modifications in staining, include:
Research specific stain using periodic acid-Schiff, Coleman's Feulgen
solution, Mayer's hematoxylin, and methylene blue in order to reduce
distortions in a 1997 published study containing Helicobacter pylori,
pathogenic GI bacteria
Contrast-enhancing dye composed of osmium tetroxide stain along
with excess thiocarbohydrazide (OTO) for lipid-containing membranes
(1966)
Double immunoenzymatic staining methods involving alkaline
phosphates and peroxidases to shade developing enzymes (2012)
New staining technique for fungal-infected plant tissues using cotton
blue and saphranin to identify embedded fungal structures in delicate
tissues (2013)
Electrophoresis staining to promote active antibody binding in tissues
to reduce time of staining (2014)

Conclusion
Staining is a very important procedure in the realm of
laboratory microbiology. It allows one to differentiate
various types of cells, both prokaryotic and eukaryotic from
each other when (a) specific dye(s) is applied. It has
evolved from the simple histological staining practices of
Anton Van Leeuwenhoek's time to the various differential
and special staining techniques of today. While not too
many entirely new stains are in existence today, It is
evident that a combination of staining techniques or
existing stains yield new methods testing for the presence
or proper visibility of target compounds.

Extra slide
While the same staining techniques are repeatedly
employed in the science of microbiology to meet current
demands, such as Gram staining and Acid-Fast staining,
as they are reliable and accurate methods for studying
microscopic specimen, new methods for staining have
been and are being developed to target specific microbes
and histological samples and their constituents. The
evolution of the differential staining procedures
mentioned above reflects the rapidly increasing need to
accommodate and expand the isolation, identification,
and study of microorganisms and their metabolism,
structures, and arrangement within their surroundings,
as well as the behavior of cells of multicellular
organisms.