Essential Microbiology Stains and Dyes Overview
Presented by
Ms. Priyanka H. Giri
Department of Microbiology
Deogiri College, Aurangabad
1
Topic :-
Stains and dyes-III
B.Sc. 1
st
yr (1
st
semester)
Paper no. 2
Microbiological techniques and
general microbiology
Ms. Priyanka H. Giri.
15/08/2020
Unit 1
Stains and Dyes.
Content:
•
Natural stains, Leucodyes, Mordants, Counter
stains, Physicochemical Basis of staining,
Fixatives and fixation of Smear. Use and
significance of stains in Microbiology, Staining
of Fungi.
Natural stains:
•
Natural stains are predominated during the early
years of bacteriology.
1)
Indigo- derived from plant indigofera and after
fermentation yield stain indigo.
2)
Indigo carmine
3)
Orcein- Obtained from lichens.
4)
Litmus- derived from livheins.
5)
Hematoxyline- Prepared by extracting log wood.
Leucodyes:
•
The chromophore group of a stain gets easily reduced
by combining with hydrogen at double bonds. This
reduction of chromophore results in loss of color.
These decolorized stains are known as Leucodyes.
•
For eg. Nitro group may be reduced to an amino group
in parasoaniline to form leuco-parasoaniline, such
satins are used as indicators of oxidation and
reduction.
•
Methylene blue reduction time (MBRT) test for
indicating quality of milk is based on the same principle
where reduction of methylene blue to leucomethylene
blue, by bacteria takes place.
Mordants:
•
A mordant may be defined as substance that
forms an insoluble compound with stain and
serves to fix the color to bacterial cell.
•
Some stains have little or no affinity for cell or it’s
components.
•
Foe eg. Cell wall, flagella, in such cases mordant is
generally applied first to the smear followed by
addition of staining solution or mordant is added
to the staining solution and the preparation is
applied to the smear in one application.
•
Compound which function as mordant are
tannic acid and salts of aluminium, iron, zinc,
copper, tin and chromium.
•
In flagella staining, tannic acid is used as
mordant.
•
Iodine in Gram’s staining is used as mordant.
Decolorizers:
•
An agent that removes the dye from the
bacterial cell is called as decolorizer.
•
Some stained cells decolorize more easily than
others depending on their chemical
composition.
•
For eg. Alcohol (Ethyl alcohol) is used in
Gram’s staining as decolorizer.
Counter stain:
•
This is also a basic dye different in color than the
initial (primary) one.
•
The purpose of counter stain is to give the
decolorized cell a color different from the first
one.
•
The microbes which are not decolorized by
alcohol retain the first stain while the decolorized
ones take up the second stain known as counter
stain.
•
For eg. In gram’s staining, saffranine is used as
counter stain.
Physicochemical basis of staining:
•
Phenomenon of staining is explained by physical and
chemical basis:
1.
Physical basis:
some dyes merely coat the surface of cell by adsorption
others dissolve or precipitate in some special parts of cell
due to osmosis, adsorption and capillarity.
The physical process is reaction between two substances
without the formation of new compound. When bacteria are
stained by physical method, there is no chemical change in
stain. Physically stained organisms loose stain when
immersed in water, alcohol or other solvent for sufficient
period of time.
2.
Chemical basis:
•
S
ome parts of cell are acidic in reaction while some other
parts are basic. According to chemical theory of staining, “
acidic
stain react with basic part of cell while basic stain react with
acidic part of stain.”
•
The process of staining involves an exchange reaction between
stain and active site of the cell.
•
For eg. The colored ions of the dye replace other ions on cellular
components.
•
Certain chemical grouping of cell protect or nucleic acid may be
involved in salt formation with positively charged ions, such as
Na+ or K+, the peripheral areas of the cell, as carrying negative
charge, in combination with positively charged ions can be
represented as (Bacterial cell-) (Na+).
•
The dye methylene blue is symbolized as
(MB+) (Cl-).
•
The ion exchange during staining will take
place, staining cell with methylene blue=
•
(Bacterial cell-) (Na+) + (MB+) (Cl-)
(Bacterial cell- MB+) +
NaCl.
Fixatives and fixation of smear:
•
Fixative is a compound used for fixation process.
•
Fixation is the process in which bacterial cell is
immobilized. This fixation can be done by two
types of fixatives i.e., physical and chemical.
•
The physical agents are heat and pressure while
chemicals are like phenol, alcohol, acid etc.
•
The fixation helps to fix the cells on the slide and
at the same time clarifies the bacterial cell
structure and their visibility and affinity of the
dye towards the cell constituents.
•
Another important role of fixatives is in killing
pathogens.
•
A good fixation process should follow the
following conditions:
i.
It should avoid the shrinkage of cell parts.
ii.
It should avoid the lysis of cell.
iii.
It should protect soluble cell substances.
iv.
The cell material should be made more rigid and
resistant.
Use and significance of stains in
Microbiology:
•
Since microbial cells are generally colorless, it is
extremely difficult to study them in unstained
preparations as structural details are not observed
clearly because of lack of density.
•
Secondly, accurate study is difficult because of the
motion of microorganisms when suspended in fluid.
•
Also, index as they fluid in which they are suspended,
which makes them nearly invisible.
•
Fixed stained smears are most frequently used hence
for the observation of morphological characteristics of
microorganisms a variety of staining methods have
been devised.
•
All these methods include certain essential steps
such as the preparation of the film or smear on
glass slide, fixation of the smear and application
of one or more staining solutions.
•
This makes the cell more clearly visible.
•
Difference between cells of different species can
also be demonstrated by differential or selective
staining which also brings out in clear details of
the various cellular characteristics or certain sub
cellular structures.
•
Stains are also used for other purpose.
•
Some stains which are also functioning as dyes
are most widely used.
•
These function as indicators and used for
determining quality of certain substances.
•
For eg. Methylene blue is most widely used as
an indicator of oxidation-reduction potential.
Staining of Fungi:
•
One of the simple stain commonly used for
staining of fungi, algae, lower bryophytes etc
is
Ehrlich’s Nematoxylin.
•
Fungi may be observed more clearly by
staining in lacto-phenol blue solution and
mounting in lacto-phenol.
This presentation delves into natural stains, leucodyes, mordants, counter stains, and their significance in microbiology. Explore the physicochemical basis of staining, fixatives, and the staining of fungi, along with examples of commonly used compounds. Gain insights into the historic use of natural stains and the role of mordants in fixing color to bacterial cells.
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Presentation Transcript
Topic :- Stains and dyes-III Presented by Ms. Priyanka H. Giri Department of Microbiology Deogiri College, Aurangabad 1
B.Sc. 1st yr (1st semester) Paper no. 2 Microbiological techniques and general microbiology Ms. Priyanka H. Giri. 15/08/2020
Unit 1 Stains and Dyes.
Content: Natural stains, Leucodyes, Mordants, Counter stains, Physicochemical Basis of staining, Fixatives and fixation of Smear. Use and significance of stains in Microbiology, Staining of Fungi.
Natural stains: Natural stains are predominated during the early years of bacteriology. 1) Indigo- derived from plant indigofera and after fermentation yield stain indigo. 2) Indigo carmine 3) Orcein- Obtained from lichens. 4) Litmus- derived from livheins. 5) Hematoxyline- Prepared by extracting log wood.
Leucodyes: The chromophore group of a stain gets easily reduced by combining with hydrogen at double bonds. This reduction of chromophore results in loss of color. These decolorized stains are known as Leucodyes. For eg. Nitro group may be reduced to an amino group in parasoaniline to form leuco-parasoaniline, such satins are used as indicators of oxidation and reduction. Methylene blue reduction time (MBRT) test for indicating quality of milk is based on the same principle where reduction of methylene blue to leucomethylene blue, by bacteria takes place.
Mordants: A mordant may be defined as substance that forms an insoluble compound with stain and serves to fix the color to bacterial cell. Some stains have little or no affinity for cell or it s components. Foe eg. Cell wall, flagella, in such cases mordant is generally applied first to the smear followed by addition of staining solution or mordant is added to the staining solution and the preparation is applied to the smear in one application.
Compound which function as mordant are tannic acid and salts of aluminium, iron, zinc, copper, tin and chromium. In flagella staining, tannic acid is used as mordant. Iodine in Gram s staining is used as mordant.
Decolorizers: An agent that removes the dye from the bacterial cell is called as decolorizer. Some stained cells decolorize more easily than others depending on their chemical composition. For eg. Alcohol (Ethyl alcohol) is used in Gram s staining as decolorizer.
Counter stain: This is also a basic dye different in color than the initial (primary) one. The purpose of counter stain is to give the decolorized cell a color different from the first one. The microbes which are not decolorized by alcohol retain the first stain while the decolorized ones take up the second stain known as counter stain. For eg. In gram s staining, saffranine is used as counter stain.
Physicochemical basis of staining: Phenomenon of staining is explained by physical and chemical basis: 1. Physical basis: some dyes merely coat the surface of cell by adsorption others dissolve or precipitate in some special parts of cell due to osmosis, adsorption and capillarity. The physical process is reaction between two substances without the formation of new compound. When bacteria are stained by physical method, there is no chemical change in stain. Physically stained organisms loose stain when immersed in water, alcohol or other solvent for sufficient period of time.
2. Chemical basis: Some parts of cell are acidic in reaction while some other parts are basic. According to chemical theory of staining, acidic stain react with basic part of cell while basic stain react with acidic part of stain. The process of staining involves an exchange reaction between stain and active site of the cell. For eg. The colored ions of the dye replace other ions on cellular components. Certain chemical grouping of cell protect or nucleic acid may be involved in salt formation with positively charged ions, such as Na+ or K+, the peripheral areas of the cell, as carrying negative charge, in combination with positively charged ions can be represented as (Bacterial cell-) (Na+).
The dye methylene blue is symbolized as (MB+) (Cl-). The ion exchange during staining will take place, staining cell with methylene blue= (Bacterial cell-) (Na+) + (MB+) (Cl-) (Bacterial cell- MB+) + NaCl.
Fixatives and fixation of smear: Fixative is a compound used for fixation process. Fixation is the process in which bacterial cell is immobilized. This fixation can be done by two types of fixatives i.e., physical and chemical. The physical agents are heat and pressure while chemicals are like phenol, alcohol, acid etc. The fixation helps to fix the cells on the slide and at the same time clarifies the bacterial cell structure and their visibility and affinity of the dye towards the cell constituents.
Another important role of fixatives is in killing pathogens. A good fixation process should follow the following conditions: i. It should avoid the shrinkage of cell parts. ii. It should avoid the lysis of cell. iii. It should protect soluble cell substances. iv. The cell material should be made more rigid and resistant.
Use and significance of stains in Microbiology: Since microbial cells are generally colorless, it is extremely difficult to study them in unstained preparations as structural details are not observed clearly because of lack of density. Secondly, accurate study is difficult because of the motion of microorganisms when suspended in fluid. Also, index as they fluid in which they are suspended, which makes them nearly invisible. Fixed stained smears are most frequently used hence for the observation of morphological characteristics of microorganisms a variety of staining methods have been devised.
All these methods include certain essential steps such as the preparation of the film or smear on glass slide, fixation of the smear and application of one or more staining solutions. This makes the cell more clearly visible. Difference between cells of different species can also be demonstrated by differential or selective staining which also brings out in clear details of the various cellular characteristics or certain sub cellular structures.
Stains are also used for other purpose. Some stains which are also functioning as dyes are most widely used. These function as indicators and used for determining quality of certain substances. For eg. Methylene blue is most widely used as an indicator of oxidation-reduction potential.
Staining of Fungi: One of the simple stain commonly used for staining of fungi, algae, lower bryophytes etc is Ehrlich s Nematoxylin. Fungi may be observed more clearly by staining in lacto-phenol blue solution and mounting in lacto-phenol.
