Staining in Microbiology

Staining
in
Microbiology
DR. MAMTA SHRIVASTAV
MD MICROBIOLOGIST

Why we need staining
Structural details of bacteria cannot be seen under light microscope due to lack of contrast. Hence, it is
necessary to use staining methods to produce colour contrast and thereby increase the visibility.

Aseptic technique
Any staining procedure should be done by using Aseptic technique.
Aseptic technique refers to the reduction of contamination from the
environment and other areas as the scientist is transferring bacteria
from one medium to another.
Aseptic techniques procedure
1. Wipe your work area before and after you are done working
2. If you ever spill something, the area needs to disinfected again with
disinfectant.
3. When dealing with microorganisms, you need to maintain sterile
conditions

Common staining techniques used in microbiology
Simple stain Negative staining Differential stain Impregnation methods
.

Basic dyes, such as
methylene blue or basic
fuchsin are used as
simple stains. they
provide the color
contrast, but impart die
same color to all
die bacteria in a smear
A drop of bacterial suspension
is mixed with dyes, such as India
ink or nigrosin. This background
gets stained black where as
unstained bacterial/yeast capsule
stand out in contrast. This is very
useful in the demonstration of
bacterial/yeast capsules which do
not take up simple stains.
Bacterial cells and
structures that are too thin
to be seen under the light
microscope, are thickened
by impregnation of silver
salts on their surface to
make them visible, e.g. for
demonstration of bacterial
flagella and spirochetes.
Two stains are used which impart different colors to different bacteria or
bacterial structures, which help in differentiating bacteria
Gram stain: It differentiates
bacteria into gram positive and
gram-negative groups
Acid-fast stain: It differentiates
bacteria into acid fast and non acid-
fast groups
Albert stain: It differentiates bacteria
having metachromatic granules from
other bacteria that do not have

SMEAR PREPARATION
A bacterial smear is a dried preparation of bacterial cells on a glass slide. Smear
preparation are made on 3× 1 glass slides .
QUALITY OF SMEAR
 It is essential that the slides be perfectly clean and free from grease, otherwise film will be uneven.
 The bacteria are evenly spread out on the slide in such a concentration that they are adequately separated
from one another, Smears should not be too thin or too thick.
 The bacteria are not washed off the slide during staining.
 Bacterial form is not distorted.
 The smear should not be more than 2-3 cm in length and should not touch the edges of the slide.
Cleaning slides
 Wipe the slide with dry cotton cloth and then holding its end with forceps, roast it free from grease by
passing it 6-12 times through a blue Bunsen flame.
 Moisten the finger with water ,rub it on surface of some fine sand soap, and then smear the surface of the
slide , after removing the soapy film with clean cloth the surface is clean and free from grease

Smear Preparation
With the wax pencil, mark the name of the bacterial culture in the far left corner on each of slides.
Liquid material
For the liquid material e.g. broth
culture, urine, sputum , pus shake
the culture tube and, with an
inoculating loop, aseptically
transfer 1 to 2 loopfuls of bacteria
to the center of the slide. Spread the
bacteria over a large area.
Solid material
When preparing a smear from a
slant or culture plate, place a loopful
of water in the center of the slide.
With the inoculating needle,
aseptically pick up a
very small amount of culture and
mix into the drop of water. Spread
this out 2-3 cm.
Allow the slide to air dry, or place it
on a slide Warmer
Pass the slide through a Bunsen
burner flame three times to heat-
fix and kill the bacteria
Smears from swabs are prepared by
rubbing it with a rolling movement
at the center of a clean glass slide
Smear from urine, body fluids or
any other fluid are prepared from
centrifuged deposits.

Fixation of smear
Fixation is the process by which the internal and external structures of cells are preserved and fixed in position.
• It also inactivates the enzymes that might disrupt cell morphology.
• It toughens cell structure so that they do not change during staining.
• It kills and fixes the cells on to the slide is done.
Fixation
Heat fixation
It is usually done for bacterial smears by gently flame
heating an air-dried film of bacteria. This adequately
preserves overall morphology but not structures within
the cells.
Method
 Allow the smear to dry completely.
 Rapidly pass the slide, smear uppermost, three times through
the flame of a burner.
 After passing the slide three times, it should be possible to lay
the slide on the back of the hand without feeling
uncomfortable. When this cannot be done too much heat has
been used.
 Allow the smear to cool before staining it.
Chemical fixation
It can be done using ethanol, acetic acid, mercuric chloride,
formaldehyde, methanol and glutaraldehyde. They are used
to protect the fin internal structure of cells. This is useful for
examination of blood smears.
method
 Allow the smear to dry in air.
 Put 1 to 2 drops of 70% alcohol and leave it for at least two
minutes or till the alcohol evaporates. Absolute alcohol may also
be used.

Hints and Precaution
 When heat-fixing a smear, always make sure that the smear is on the top of the slide as you pass it
through the flame.
 Bacteria growing on solid media tend to cling to each other and must be dispersed sufficiently by
diluting with water. If this is not done, the smear will be too thick and uneven. Be careful not to
use too much paste in making the smear. It is easy to ruin your results by using too many bacteria.
 Always wait until the slide is dry before heat-fixing.
 Fixing smears with an open flame may create artifacts.
 The inoculating loop must be relatively cool before inserting it into any broth. If the loop is too
hot, it will spatter the broth and suspend bacteria into the air. Always flame the inoculating loop
after using it and before setting it down.
 When rinsing with water, direct the stream of water so that it runs gently over the smear.

Gram stains
This staining technique was originally developed by Hans Christian Gram (1884). Later modification gives
better results.
Purpose Principle of the method Type of primary sample
Visualization of microbes
in clinical samples or from
culture plates and their
classification as either gram
positive , gram negative
and few species gram
variable
Gram-positive bacteria retain the primary
stain (crystal violet) by resisting
decolorization appear purple. Gram-negative
bacteria on the action with acetone thus other
hand get completely decolorized and take up
the colour of the counter stain safranin and
appear light red or pink.
Pus, Throat Swab, Body
fluids, Culture Isolates,
Sputum, Wound Swab,
Eye Swab etc.

Gram stain
A Crystal violet or methyl violet (0.5-2%)
Absolute Alcohol (100% ethanol)
Distilled water
10 g
100 ml
1000 ml
B Grams Iodine:
Iodine Crystal
Potassium Iodine
Distilled Water
10 g
20 gm
1000 ml
C Decolourizer :
Acetone or absolute alcohol or acetone alcohol or Iodine
acetone
D Counter Stain
Safranin containing 99% dye
Distilled Water
5 g
1000 ml
Equipments and reagents:
Glass Slides, Burner, Wire loop, Microscope, Staining Rack and Stains.
Gram stain is commercially available ; however it can be prepared in the laboratory as follows..

Gram staining procedure
 Fixation: The smear made on a slide from bacterial culture or specimen, is air dried and then heat
fixed.
 Primary' stain: The smear is stained with crystal violet (or gentian violet or methyl violet) for one
minute. Then the slide is rinsed with water. Crystal violet stains all the bacteria violet in color
(irrespective of whether they are gram-positive or negative).
 Mordant: Gram's iodine (dilute solution of iodine) is poured oven he slide for one minute. Then the
slide is rinsed with water. Gram's iodine acts as a mordant.
 Decolorization : Next step is pouring of few drops of decolorizer to the smear: e.g. acetone (for 1-2
sec) or ethanol (20-30 sec) or acetone alcohol (for 10 sec) or iodine acetone. Slide is immediately rinsed
with water.
o Decolorizer removes the primary stain from gram negative bacteria while the gram-positive
bacteria retain the primary stain.
o Note: Decolorization is the most crucial step of Gram stain. If the decolorizer is poured for more time,
even gram positive bacteria loose color (over decolorization) and if poured for less time, the
gram·negative bacteria do not lose the color of primary stain properly (under decolorization).
 Counter stain: Secondary stains such as safranin or diluted carbol fuchsin is added for 30 seconds. It
imparts pink or red color to the gram-negative bacteria. Aternatively, neutral red may also be used as
counter stain, especially for gonococci. The slide is rinsed in tap water, dried, and then examined under
oil immersion objective.
 Interpretation : Gram positive bacteria resist decolorization and retain the color of primary stain
and appear violet. Gram negative bacteria are decolorized and therefore take counterstain and
appear pink.

Mechanism of Gram stain and Structure of bacterial cell wall
 The violet dye and iodine combine to form an insoluble, dark
purple compound in the bacterial protoplasm and cell wall.
This compound is dissociable in the dicolouizer, which
dissolves and removes its two components from the cell.
 But the removal is much slower from Gram positive than
Gram negative bacteria, so that by correct timing the former
stay dark purple while the letter become colourless.
 The difference between the two types of bacteria is that gram
positive have thicker and denser peptidoglycan layers in their
cell walls, which makes them less permeable to the strain than
those of the Gram negative bacteria.
 The iodine has a critical role in enhancing this difference. It
seems to bind temporarily to the peptidoglycan and make it
even less permeable to the dye.

Quality control and bacteria under the Microscope
.
Quality control of gram strain
Gram positive – S. aureus ATCC25923
Gram negative – E. Coli ATCC 25922.
Interpretation of results:
Purple bacteria – gram positive.
Pink to Red bacteria – gram negative
Gram variable
Different Morphology of bacteria seen in gram stain

Modification of Gram stain
 Kopeloff and Beerman’s modification : Primary stain and
counter stain used are methyl violet and basic fuchsin
respectively.
 Jensen’s modification: This method involve use of absolute
alcohol as decolorizer and neutral red as counter stain. It is useful
for meningococci and gonococci.
 Weigert’s modification : This modification is useful for staining
tissue sections. Here, aniline – xylol is used as a decolorizer.
 Preston and Morrell’s modification: Here, iodine- acetone is
used as decolrizer.

Automated Gram Stainer
A market-leading automated Gram Stainer
PREVI® COLOR sprays
•Standardization – slides are all stained the same
way
•Up to 30 slides ready in 5 minutes
•Eco-friendly system
•Staining adjustable to your practices/habits

Uses of Gram Stain
 Differentiation of bacteria into gram-positive and gram-negative:
 For identification: Gram staining from bacterial culture gives an idea to put the
corresponding biochemical test for further identification of bacteria
 To start empircal treatment: Gram stain from specimen gives a preliminary
clue about the bacteria present (based on the shape and Gram staining property
of the bacteria) so that the empirical treatment with broad spectrum antib iotics
can be started early b efore the culture report is available.
 For fastidious organisms, such as Haemophilus which takes time to grow in
culture; Gram stain helps in early presumptive identification .
 Anaerobic organisms, such as Clostridium, which do not grow in routine
culture. Hence, Gram stain gives a preliminary clue to put anaerobic culture.
 Yeasts: In addition to stain the bacteria, Gram stain is useful for staining
certain fungi such as Candida and Cryptococcus (appear gram-positive).

Ziehl-Neelsen Stain (ZN STAIN)

ZN STAIN
The acid-fast stain was discovered by Paul Ehrlich and subsequently modified by Ziehl and Neelsen.
Purpose Principle Sample

This staining is done co identify acid-
fast organisms, such as Mycobacterium
tuberculosis , M. lepare etc. Acid
fastness is due to presence of mycolic
acid in the cell wall.
Acid fast bacilli e.g. Mycobacterium spp resist
decolorisation by acid after they have been stained
with a strong phenolic dye like carbol fuchsin by
virtue of their unique cell wall stricture. Sulphuric
or hydrochloric acid may be used for
decolorisation and methylene blue or malachite
green as counter stain
To detect acid fast bacilli in
different samples e.g. sputum,
urine, CSF, pus, BAL fluid, other
body fluids, nasal smear, slit skin
smear, tissue etc.
Container: Sterile plastic container. (slit skin smears and nasal smears are collected directly onto a glass slide).
Specimens that cannot be processed within 1 hour of the time of collection should be refrigerated during transport to the
laboratory. Sputum – Early morning sample is best.

ZN STAIN
Mycolic acid in the cell wall is responsible for acid fastness
 Mycolic acid confers very low permeability to the cell wall and is responsible for acid fastness.
 Concentration of sulphuric acid may vary depending on the acid fastness of the structure to be demonstrated .
More the content of mycolic acid in cell wall, more is the acid fastness, hence more is the percentage of
sulphuric acid needed for decolorization.
Cell wall of acid fast bacteria

ZN STAIN
Equipment and Reagents:
Glass Slides, Burner, Wire loop, Microscope, Staining Rack and Stains.
Z.N. Stain is commercially available; however it can be prepared by this reagents in the laboratory as follows.
ZN Reagents Quantity
a) ZN Carbol fuchsin
Basic fuchsin 5 g
Phenol (Crystalline) 25 g
Alcohol (95 % or 100% ethanol) 50 ml
Distilled water 500 ml
b) Sulphuric acid (20%) decolourizer
Concentrated sulphuric acid (98%) 250 ml
NOTE: The acid must be added to the water ; it is dangerous to add water to the acid
c)Alcohal 95%
Ethanol 95 ml plus water to 100 ml, or industrial methylated spirit
d)Methylene blue counterstain
Methylene blue 5 g

Ziehl-Neelsen Technique (Hot Method)
Preparation of smear
Smears (2x3 cm size) are prepared from thick mucopurulent part of sputum
Smears from swabs are prepared by rubbing it with a rolling movement at the center of a clean glass slide.
The smear should not be more than 2-3 cm in length and should not touch the edges of the slide.
Smear from urine, body fluids or any other fluid are prepared from centrifuged deposits. Smears should not be too thin or too thick.
fixation
The smear is heat fixed.
Primary stain
Cover the slide with ZN carbol fuchsin filtered through Whatman No. 1 paper. for 5 minutes with intermittent heating by flaming the
underneath of the slide until the fumes appear. Heating helps in better penetration of the strain. Do not allow the strain to dry on the
slide. Heating act as mordant here.
Decolorization
Cover the slide with 20% sulfuric acid after wash with water. Decolourization generally requires contact with sulpuric
acid for a total time of at least 10 min. When it is complete, the film, after washing , is only very faintly pink.
Counterstain
It is done by adding methylene blue for 1 minute after wash with water. Slide is rinsed
in tap water, dried, and then examined under oil immersion objective.
Interpretation
Acid fast bacilli are stained bright red/pink coloured acid-fast while other non-acid fast organisms present in
the smear and the background take up the counter stain and appear blue

Acid fast bacteria under microscope
 GRADING OF POSTIVE SMEARS :
RNTCP GRADING
Mycobacterium tuberculosis
appers long, slender,beaded,
red coloured acid fast
bacillilus
Grading for smear of
Mycobacterium leprae red
acid bacilli , arranged
singly or in groups(cigar
like bundles/ globi
No. of bacilli per oil immersion field (OIF) Grading
1-10 bacilli in 100 OI 1+
1-10 bacilli in 10 OIF 2+
1-10 bacilli per OIF 3+
>1000 bacilli or bacilli in clumbs and globi in each OIF 6+

Modifications of Acid Fast Staining
Modifications are as follows:
 Cold method (Kinyoun's method): It is differs from ZN staining in that
 It is modification, where the intermittent heating is not required.
 Phenol concentration in carbol fuchsin is increased,
 Duration of carbol fuchsin staining is more.
 Example : Nocardia spp.
 Acid-alcohol can be used as decolorizer alternatively.
 Acid alcohol (3% hydrochloric acid+ 95% ethyl alcohol): It can be used to differentiate M. tuberculosis (acid and
alcohol fast) from M. smegmatis(acid fast but not alcohol fast) in urine sample.
Nocardia SPP: Nocardiae are partially acid fast
and appear as branching and filamentous red
colored acid fast bacilli 1% sulfuric acid use as
decolorizer

Auramine Phenol Technique
It is a florescent staining technique, uses auramine phenol as primary stain, acid alcohol as decolorizer
and potassium permanganate as counter stain.
- The bacilli appear bright brilliant green against dark background.
- Smears screened by using 20 x objective , hence can be screened faster (2 min for 100 fields)
- It is widely used bt RNTCP in laboratory having higher sample load
Tubercle bacilli seen under fluorescence microscope

ALBERT STAIN
Corynebacterium diphtheriae

ALBERT STAIN
Albert stain is used to demonstrate the metachromatic granules of Corynebacterium diphtheriae.
Procedure
 Fixation: The smear is heat fixed.
 Smear is covered with Albert 1 (Albert's stain) for 5 minutes, then is washed in water, and blotted
dry.
 Albert ll (iodine solution) is added for 1 minute.
 Slide is washed in water, blotted dry and examined under oil immersion field.
 Interpretation
C. diphtheriae appears as green colored bacilli arranged in Chinese
letter or cuneiform pattern, with bluish black metachromatic
granules at polar ends. These can be differentiated from
diphtheroids which do not show granules and are arranged in
palisade pattern.

Microscopy of bacteria in living state
Unstained (Wet) Preparations :
Unstained preparations are examined mainly for checking bacterial motility (e.g. hanging drop and
wet mount preparations) and for demonstration of spirochetes (e.g. dark field or phase contrast
microscopy).
Vital Stains :
Vital stains are capable of differentiating the living cells from dead cells. The live cells are capable of
excluding the dye and stain negatively whereas the dead cells stain positively as they cannot exlude
the dye. the viability can be assessed by counting the percentage of total cells that stain negatively.
Vital stains have greater applications in some diagnostic and surgical techniques.
In supravital staining, living cells that have been removed from an organism are stained (in
vitro), whereas the intravital staining is done by injecting stain into the body (in vivo).
Examples of vital stains are eosin, propidium iodide, trypan blue, erythrosine and neutral red.

Staining use for fungal disease
1) Potassium hydroxide (KOH) preparation: Keratinized tissue specimens such as skin scrapings and plucked
hair samples are treated with 10% KOH which digests die keratin material so that the fungal hyphae will be
clearly seen under the microscope.
 Concentration of KOH
10% KOH – most commomly use
20-40% KOH – specimens takes longer times to dissolve example. Nail scraping, biopsy tissues.
 Glycerol (10%) is added to prevent drying
 DMSO ( dimethyl sulfoxide) is added which helps in tissue digestion.
2) Gram stain: lt is useful in identifying the yeasts (e.g. Cryptococcus ) and yeast like fungi (e.g. Candida). They
appear as gram-positive budding yeast cells.
KOH mount Gram stain cryptococoocus Gram stain candida

3) India ink and nigrosin stains: They are used as negative stains for demonstration of capsule of Cryptococcus neoformans.
4) Lactophenol cotton blue (LPCB): It is used to Study the microscopic appearance of the fungal isolates grown in
culture.
It contains:
• Phenol acts as disinfectant.
• Lactic acid preserves the morphology of fungi.
• Glycerol prevents drying.
• Cotton blue stains the fungal elements blue.
5) Calcofluor white stain: lt is more sensitive than other stains; binds to cellulose and chitin of fungal cell wall and fluoresce
under UV light.
India ink nigrosin stain cryptococcus

6) Histopathological stains: They are useful for demonstrating fungal elements from biopsy tissues. This is useful for
detecting fungi causing deep mycoses.
a) Periodic acid Schiff (PAS) stain: lt is the recommended stain for detecting fungi. PAS positive fungi appear magenta/deep
pink, whereas die nuclei stain blue.
b) Gomori methenamine silver (CMS) stain: 1t is used as an alternative to PAS for detecting fungi. lt stains both live and dead
fungi, as compared to PAS which stains only the live fungi. GMS stains the polysaccharide component of !he cell wall. Fungi
appear black whereas the background tissue takes pale green color.
c) Mucicarmine stain: lt is used for staining die carminophilic cell wall of Cryptococcus and Rhinosporidium.
d) Masson fontana stain: lt is used for pigmemed (or pheoid) fungi.
PAS stain shows septate fungal hyphae

Stains use for Parasitology
1) Direct Wet Mount(Saline and Iodine Mount): Drops of saline and Lugol’s Iodine are placed on two corners
of slide. A small amount of feces is mixed by a stick to form a uniform smooth suspension. Cover slip is placed
on the mount and examined under low power objective(10 X) followed by high power objective (40 X).
Parasites Eggs/Larvae seen in wet preparation

Stains use for Parasitology
2) Parmanent Stained Smear: Permanent stained smears are required for accurate diagnosis of
intestinal parasites.
 Iron hematoxyline stain
 Trichrome stain
 Modified acid fast stain
3) Romanowsky’s stain for Thick and thin blood smear
 Water based stains- Geimsa stain, Jaswant singh bhattacharya (JSB) , Field stains
 Methanol based stains- Leishman’s stain and wright’s stain.
Modified acid fast stain:Cryptosporidium spp Isospora

Staining in Microbiology

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