General Microbiology PTU - Chapter 1
Unit 1.
Staining methods
1.0 Introduction
Staining of bacteria is of primary
importance in the study of bacteria. Staining is needed because the bacteria
are nearly transparent under the light microscope. Differential stains impart
different colors to different types of bacteria helping in classifying them in
various classes. Special staining methods are used to demonstrate the different
orgenells which make up a bacterium.
1.1 Unit
objectives
After studying this unit student would
be able to know:-
-How to prepare the smears and tissue
sections for staining.
-Different
types of staining methods.
-Principles
behind theses methods.
-Preparation
of reagents for various staining methods.
-Special
stains and their uses.
1.2 Preparation of smears:-
Before learning how to stain the smears it is important to learn how to prepare
a good smear. Films are prepared on glass slides or coverslips, mostly on glass
slides.
1.2.1 Cleaning of glass slides
and coverslips:-
-Glass slides and coverslips
which are ready to use (i.e. certified clean and grease free) can be obtained
from the manufacturers.
-If the glass slides are not
‘ready for use’ they should be wiped with a clean cotton cloth and then passed
through a Bunsen flame 6-12 times. This removes all the grease.
-The slide should be allowed to
cool before keeping it down to avoid cracking.
-A drop of water can be spread in
a thin even film on a perfectly clean glass slide.
-If it is not clean the water
would collect in small drops and a film cannot be made.
-Used glass slides should be
discarded. They should not be reused because the removal of all the organisms
with washing cannot be ensured.
-Coverslips if used should not be
more than 0.1 mm. thick.
-They are cleaned by immersing
for several days in a solution of concentrated sulphuric acid saturated with
potassium dichromate kept at room temperature in a glass jar.
-They are then washed with
distilled water and stored in a stoppered jar containing 50% ethyl alcohol.
-They are wiped with a dry cotton
cloth before use.
1.2.2 Making films:-
-If a fluid material such as
urine, pus, sputum, culture broth is used for preparing the film, then a
loopful is taken and spread over the slide.
-A film so produced would have
both thin and thick areas, which can be advantageous, as shall be learned with
practice.
-If a solid material like a
colony grown on agar is used to make a film a loopful of water is first taken
on the slide.
-To it is added a minute quantity
of the material obtained by just touching the growth.
-This is then emulsified in the
water and spread as a thin film over the slide.
-The smear so prepared is dried
by holding it high over a Bunsen flame.
-The dried smear is then ‘fixed’
by slowly passing the smear with the dried side downwards through the flame
three times.
-Instead the smear can be fixed
by holding the slide with film upwards over the top of the Bunsen flame for a
few seconds. The slide should not be overheated.
-When the slide becomes just too
hot to be touched on the back of the hand the fixation is completed.
-For preparing films (as well as
for staining them) on a coverslip, Cornet’s forceps are used which hold the
coverslip in a horizontal position, when the forceps are kept on the bench.
1.2.3 Marking of films:-
-If a slide with a matt surface
at one end is used it can be marked on that surface using an ordinary graphite
pencil.
-Otherwise a marking diamond of a
grease pencil is used to write on one side of the slide.
-Stick-on labels should not be
used. They are liable to be defaced or washed off during staining process.
1.2.4 Staining:- Both films as
well as tissue sections may need to be stained. The procedure used for them
are:-
(a) Films:- the stain is poured
over the film covering its entire area.
-The amount of stain should be
sufficient to ensure that it doesn’t evaporate during staining.
-The area bearing the
identification mark should not be covered by the stain.
-When staining procedure is over
the slide is washed with water and allowed to dry in a vertical position.
-It is then observed under
oil-immersion objective.
(b) Tissue sections:- The
sections are embedded in paraffin which is impervious to water. To allow the
watery stains to penetrate it has to be removed.
-Paraffin is removed by keeping
the slide bearing the section in a jar of xylol for some minutes.
-The section is then treated with
a few drops of absolute (100%) ethyl alcohol. The section should turn opaque.
-Then a few drops of 50% alcohol
are poured on the slide.
-It is then washed in water.
-The section is then stained with
the stain required.
-After staining the section is
washed with water and the excess of the water is removed by pressing the slide
between fluffless blotting paper.
-Immediately few drops of 95%
alcohol are poured over the section.
-This is followed by pouring few
drops of absolute alcohol.
-The slide is then immersed in a
jar of xylol.
-When the section becomes clear
in appearance it is removed from the xylol.
-The excess of xylol is wiped
away and the section is mounted with a No.1 (1 mm. thick) coverslip using a
drop of Canada balsam.
-It is then observed under
oil-immersion objective.
-The section should not be
allowed to dry through this entire procedure.
1.3 Simple stains:-These stains are
used to demonstrate the presence of organisms in the clinical sample.
(a) Loeffler’s
Methylene blue.
It
is the most commonly used simple stain.
It
is prepared as follows:-
Saturated
solution of Methylene blue in absolute alcohol – 300 ml.
KOH
0.01% in water – 1000 ml. is added to the above and used.
Procedure:-
Pour the stain over the film. Keep for 3 minutes. Wash with tap water.
(b)
Polychrome Methylene blue.
It
is slowly ‘ripened’ Loeffler’s methylene blue. The ‘ripening’ is achieved by
keeping the Loeffler’s methylene blue in bottles which are half filled. These
bottles are shaken periodically so as to aerate the stain. The oxidation of
methylene blue leads to the formation of a violet compound which gives the
stain its polychrome property. It may take up to a year for the stain to ripen.
The ripening process can be quickened by adding 1% Potassium carbonate to it.
The
procedure for its use is similar to Loeffler’s Methylene blue.
(c)
Dilute Carbol fuschin.
It
is used rarely because overstaining with it can occur easily.
It
is prepared as follows:-
Dilute
the Strong Carbol fuschin (used in Ziehl-Neelsen’s stain) 10 to 15 times using
distilled water.
Procedure:-
Pour the stain over the film. Keep for 10-25 seconds. Wash with tap water.
1.4 Negative Staining.
It is called ‘negative staining’
because here the background is stained, while the bacteria are not. India ink
preparation is mixed on a glass slide with the culture suspension or the
material containing bacteria. A thin film of this mixture is prepared using a
loop, which is allowed to dry before examining. The capsulated bacteria or
spirochaetes are seen as clear transparent objects on a dark-brown background.
Check your progress:-
State true or false.
1) A loopful of water shall form
small separate water drops on a perfectly clean glass slide.
2) Cornet’s forceps are used
while preparing and staining of smears on coverslips.
3) Stick-on labels are best for
marking the smears.
4) Tissue sections should be
dried before staining with watery stains.
5) Polychrome Methylene blue is
ripened Loffeler’s methylene blue.
6) Negative staining stains only
the background and not the bacteria.
1.5 Differential staining.
These methods are used to
differentiate between different types of bacteria based on the colors they take
during these staining procedures.
a)Gram’s Stain.
This is the most commonly
performed staining procedure in any microbiology laboratory. It differentiates
the bacteria into two groups, the Gram Positive and the Gram Negative bacteria.
This differentiation is clinically very important because the two groups are
sensitive to different types of antibiotics. This is also the reason for it to
be the most often used staining method in a microbiology laboratory.
Principle of Gram’s stain:- When
a basic para-rosaline dye such as Crystal violet (or Methyl violet) is used to
stain certain type of bacteria, it can be ‘fixed’ inside the bacteria by adding
Iodine after the para-rosaline dye. In these bacteria the subsequent treatment
with a decolorising agent such as acetone (or alcohol) doesn’t remove the dye.
In
other type of bacteria no such ‘fixing’ is observed and the addition of a
decolorising agent removes both the para-rosaline dye and the iodine, rendering
such bacteria colorless. These bacteria are then counterstained by a
contrasting dye (usually red) to demonstrate them easily.
The group of bacteria which
retain the para-rosaline dye even after application of decolorizing agent (i.e.
appear violet) is called “Gram Positive bacteria”, while the group that loses
the stain (i.e. appear red) is called “Gram Negative bacteria.”
Various theories have been put
forward to explain the Gram reaction. The most accepted theory is based on the
presence of Magnesium ions in gram positive bacteria’s cell wall. These
magnesium ions are supposed to be responsible for the formation of the methyl
violet-iodine complex in the cell walls of gram positive bacteria. Since gram
negative bacteria lack magnesium ions in their cell walls they are not able to
form this complex. Therefore when a decolorizing agent is applied the unbound
methyl violet and iodine are washed away from a gram negative cell wall, while
in the gram positive cell wall the complex formed by them remains attached to
the cell wall.
The methodology of Gram’s stain
has been modified by several scientists. Two methods of Gram’s stain are
generally used. They are:-
1)Kopeloff and Beerman’s method.
This method uses acetone as a
decoloriser .
Reagent preparation:-
Methyl violet:- dissolve 10 grams
of Methyl violet 6B in 1 litre of distilled water.
Iodine solution:- dissolve 20
grams of Iodine in 100 ml. of 4% NaOH. Once it is dissolved completely add 900
ml. of distilled water to it, to make upto 1 litre.
Acetone:- use 100% acetone
Basic fuchsin:- dissolve 0.5 gram
of Basic fuchsin in 1 litre of distilled water.
Procedure:-
-Make a smear of the material to
be stained on a clean glass slide.
-Fix the smear by holding it,
film upwards, above the Bunsen flame for a few seconds. The fixation is
complete when the slide is just too hot to touch on the back of the hand.
-Cover the whole slide with
methyl violet and keep it for 5 minutes.
-Tip off the methyl violet. Wash
the slide with Iodine while holding it at a slope, from top to bottom. Remove
all the crystalline deposit.
-Cover the whole slide with
Iodine and keep for 2 minutes.
-Decolorise with Acetone. Tip off
all the iodine, then hold the slide at a slope. Keep the tap water
running. Pour acetone from top to bottom
for 3 seconds. Immediately wash in running tap water. Over decolorisation may
render Gram positive bacteria to appear as Gram negative, hence the timing of
contact with acetone is very important.
-Cover the slide with basic
fuchsin and keep for 30 seconds.
-Wash thoroughly in tap water and
let the smear air dry.
-Examine under Oil immersion
objective.
2) Jensen’s modification of
Gram’s stain.
This method uses alcohol as a
decoloriser which is slower in action. It is also less time consuming. It can
be recommended for routine bacteriological work.
Reagent preparation:-
Methyl violet:- dissolve 5 grams of methyl violet 6B
in 1 litre of distilled water.
Iodine solution:- dissolve 20
grams 0f potassium iodide in 250 ml. of distilled water.
Then add 10 grams of iodine. When
dissolved make upto 1 litre with distilled water.
Alcohol:- use 100% ethyl alcohol.
Neutral red:- add 1 gram of
neutral red and 2ml. of 1% acetic acid
to 1 litre of distilled water.
Procedure:-
-Make the films, dry and fix as
stated above.
-Cover the whole slide with
methyl violet and keep for 30 seconds.
-Tip off the stain. Wash the
slide with iodine while holding it at a slope.
-Cover the slide with fresh
iodine and keep for 30 seconds.
-Wash off the iodine with
absolute alcohol.
-Cover the slide with alcohol and
keep tilting it from side to side for 10 to 15 seconds (The optimum timing for
decolorizing may vary from lot to lot).
-Wash with water.
-Cover the whole slide with
neutral red and keep for 1 minute.
-Wash thoroughly with water and
let the smear to air dry.
-Examine under oil-immersion
objective.
The gram positive bacteria would appear
violet while the gram negative would appear red in color. To ensure that
staining has been proper a film of pure culture of Staphylococcus aureus may be
made on one side of the smear to be stained. It should stain violet in color
for the procedure to called satisfactory.
There are many other
modifications of Gram’s stain. In his original method the smears were stained
with aniline-gentian violet and treated with Lugol’s iodine. Alcohol was used
as a decoloriser, while
b) Acid fast bacilli staining.
Acid fast bacilli are those which
once treated with aniline dyes, resist decolorisation (therefore called
color-fast) even in the presence of a strong acid. The genus Mycobacterium
which contains the causative organisms of tuberculosis and leprosy is prime
example of acid fast bacteria.
Mycolic acid which is found in
the cell wall of Mycobacteria is supposed to impart the acid fast property to
them. The staining methods used to
demonstrate acid fast bacilli are:-
Ziehl-Neelsen Staining
It was originally described by
Ehrlich and was modified by Ziehl and Neelsen.
Reagent preparation:-
Ziehl-Neelsen’s (strong) carbol
fuchsin:- dissolve 5 gm. of basic fuchsin in 25 gm. of crystalline phenol by
keeping them in a 1 litre flask over a boiling water bath, for 5 minutes,
shaking the flask from time to time. When there is complete solution add 50 ml.
of absolute alcohol and mix thoroughly. Then add 500 ml. of distilled water to
it. Filter it before use.
20% sulphuric acid solution:-
Take 800 ml. of distilled water in a large flask. Add 200 ml. of concentrated
sulphuric acid to it by slowly pouring it down the side of the flask into the
water. About 50 ml. of acid should be poured (slowly) at a time. The mixture
will become hot. It should be mixed gently and the remainder of the acid poured
similarly. Warning:- Never pour water into acid. It is dangerous. The heat of
the reaction would cause the water to boil and it may lead to serious burns.
Loeffler’s methylene blue:- see
above.
Procedure:-
-Prepare and fix the films as
described above, using only fresh slides.
Keep the smear on a stand that allows access to the slide’s bottom.
-Cover the slide with strong
carbol fuchsin and heat it from below till steam rises from the carbol fuchsin
solution. Remove the burning torch (twist a small piece of cotton on the tip of
an inoculating wire, dip it in spirit and light it to make a burning torch) and
reheat when steam stops coming. Don’t allow the stain to boil. Remove the
burning torch as soon as first wisps of steam are seen. Don’t allow the stain
to dry. Add more stain to keep the slide covered. Allow the smear to stain for
5 minutes.
-Wash with water.
-Cover the slide with 20%
sulphuric acid. The red color would change to yellowish brown. After 1 minute
wash the slide with tap water and again cover it with 20% sulphuric acid.
Repeat this process twice or thrice more, allowing a total of 7 to 10 minutes
of contact time with sulphuric acid.
-Wash the slide in tap water.
-Cover the slide with Loeffler’s
methylene blue for 30 seconds.
-Wash with tap water and allow the
smear to air dry.
-Examine under oil-immersion
objective.
Acid fast bacilli would appear
bright red while the surrounding tissue cells as well as other bacteria would
appear blue. If the tissue cells appear red then the smear was not decolorized
properly. A fresh smear should be prepared and stained in such a case. The
slides should be discarded after use and not recycled.
Instead of Loeffler’s methylene
blue Malachite green too can be used (the background would appear green instead
of blue). Some people use 95% alcohol as a secondary decoloriser. It is added
as an extra step after decolorizing with sulphuric acid. Instead of 20%
sulphuric acid one can use ‘acid-alcohol’ as a decoloriser. It consists of 3
ml. of concentrated hydrochloric acid in 97 ml. of 95% alcohol.
Modifications of Ziehl-Neelsen’s
method:-
The above method is used for
demonstration of Mycobacterium tuberculosis.
Mycobacterium leprae is also acid
fast but to a lesser degree than tubercle bacillus. Thus for demonstrating
lepra bacilli 5% sulphuric acid is used.
Similarly ‘clubs’ caused by Actinomycetes
and Nocardia are acid fast when treated with 1% sulphuric acid.
Cultures of some Nocardia species
are acid fast when decolorized with 0.5% sulphuric acid.
Most bacterial spores are also
acid fast.
c)Fluorochrome staining.
Here the fluorescent dye Auramine
O is used instead of Carbol fuchsin. While no heating is involved this
procedure requires a fluorescent microscope. It is a costlier but much easier
and rapid method.
d)Albert’s stain.
Volutin (polyphosphate) granules
are found in the cytoplasm of Diphtheria bacillus. They appear as refractile
round bodies in bacterial cytoplasm in wet preparetions. With Toludineblue or
methylene blue they stain metachromatically, appearing reddish-purple with the
rest of the cytoplasm of the bacteria taking the color of the counterstain
(green).
Reagent preparation:-
Albert’s Staining solution:-
dissolve 1.5 gm. of toludine blue and 2 gm. of malachite green in 20 ml. of 95%
ethyl alcohol. Add this mixture to 1 litre of distilled water containing 10 ml.
of glacial acetic acid.
Albert’s iodine:- dissolve 6 gm.
of iodine and 9 gm. of potassium iodide in 900ml. of distilled water.
Procedure:-
-Prepare, dry and fix the smear.
-Cover the slide with Albert’s
staining solution and keep it for 5 minutes.
-Wash in tap water.
-Cover the slide in Albert’s
iodine and keep for 1 minute
-Wash in tap water, allow to air
dry.-Examine under oil-immersion objective. The granules stain bluish-black,
the rest of the protoplasm appears green, while other cells and organisms
appear light green.
1.5Cytological staining
a) Staining of capsules:-
capsules of bacteria present in animal tissues, blood, pus, fluids etc. are
clearly demonstrated by the commonly used stains. However when artificial
cultures of bacteria (grown in the laboratory on media) are examined, special
methods must be employed for their demonstration. ‘Negative staining’ using
India ink is a preferred method. Wet films are generally preferred to the dry
India ink method described above. Care should be taken to press the cover slip
on the ink drop while it is covered in a blotting paper, so that the film
becomes very thin and pale in colour.
The bacterial cell appears as a
refractile body with a clear zone around it, abutted by the ink. In
non-capsulated bacteria no such zone is seen. ‘Positive capsular stains’ are
not reliable and not recommended.
b) Staining of flagella:- Because
of their extreme thinness they are best demonstrated with electron microscope.
To demonstrate them under light microscope their thickness has to be increased
at least 10-fold by superficial deposition of stain. The stain used is basic
fuchsin with tannic acid. It is deposited on the flagella and the bacteria from
an evaporating alcoholic solution.
c) Staining of intracellular
lipid:-
d) Staining for bacterial
nuclei:- Bacterial nucleus can be differentiated from the cytoplasm by using
Robinow’s method. The cells are first treated `with 1mol/litre of HCl at 60oC,
and then stained with Giemsa’s stain. The nuclei appear dark purple while the
cytoplasm is very lightly stained.
e) Staining for bacterial
spores:- bacterial spores are not stained by ordinary stains like Gram’s stain.
In spore bearing organisms they appear as a clear area in the cytoplasm of the
bacterium. As described above modified Ziehl-Neelsen method can be used to
stain them.
Check
your progress:-
Fill
in the blanks-
1)
In a gram’s stained smear the Gram Positive organisms appear ___________ in
color.
2)
The gram’s staining property is due to the presence of ____________ ions in the
cell wall of Gram positive bacteria.
3)
Acetone is used as a ________________ agent in gram’s stain.
4)
_____________ is responsible for acid fastness of Mycobacterium species.
5)
In Albert’s stain the metachromatic granules appear _________________ in color.
6)
The Mycobacterium tuberculosis bacteria are acid fast up to __________ of
sulphuric acid, while Mycobacterium leprae are acid fast up to ________ of
sulphuric acid.
1.6 Summary.
Different types of stains and
staining methods are utilized in microbiology. The simple stains are used to
demonstrate the presence of bacteria. Differential stains like the Gram’s stain
and Z.N. stain are used to differentiate between the bacteria based on their
staining property. This differentiation is clinically very useful in rapidly
indicating the choice of therapy to be given to the patient. Special staining
methods are required to demonstrate the various orgenells of the bacteria, they
are rarely used in routine microbiology work.
1.7 Answers to check your
progress:-
State
true or false:- 1) False, 2) True, 3)False, 4) False, 5) True, 6) True.
Fill
in the blanks:- 1) Violet. 2) Magnesium. 3) Decoloriser. 4) Mycolic acid. 5)
Reddish purple. 6) 20%, 5%.
1.8
Exercise
Q.1
Describe the procedure of cleaning the glass slides and coverslips and the
making of films on them.
Q.2
Enumerate the simple stains. What is the difference between simple and
differential stains?
Q.3
What is the importance of Gram’s stain? Describe the procedure of staining a
smear by Jensen’s modification of Gram’s stain.
Q.4
What do you understand by acid fastness of a bacteria? Describe the procedure
of Ziehl Neelsen staining for Mycobacterium tuberculosis.
Q.5
What are metachromatic granules? Describe the procedure of the staining method
used to demonstrate them.
Q.6
Write short notes on:-
(a)
Preparation of tissue sections for bacterial staining procedures.
(b)
Preparation of 20% sulphuric acid solution.
(c)
Capsular staining.
(d)
Staining of spores.
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