staining method

1. Staining methods

2. • Staining is a technique used to enhance contrast in
samples, generally at the microscopic level.
• Stains and dyes are frequently used in histology (the
study of tissue under the microscope) and in
the medical fields of histopathology, hematology,
and cytopathology that focus on the study
and diagnoses disease at a 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.
• In vivo staining (also called vital staining or intravital
staining) is the process of dyeing living tissues. By
causing certain cells or structures to take on contrasting
colour(s), their form (morphology) or position within a
cell or tissue can be readily seen and studied.

3. • The usual purpose is to reveal cytological details that might
otherwise not be apparent; however, staining can also reveal
where certain chemicals or specific chemical reactions are
taking place within cells or tissues.
• In vitro staining involves colouring cells or structures that
have been removed from their biological context. Certain
stains are often combined to reveal more details and features
than a single stain alone.
• Combined with specific protocols for fixation and sample
preparation, scientists and physicians can use these standard
techniques as consistent, repeatable diagnostic tools.
A counterstain is stain that makes cells or structures more
visible, when not completely visible with the principal stain.
• For example, crystal violet stains only Gram-positive bacteria
in Gram staining. A safranin counterstain is applied that stains
all cells, allowing identification of Gram-negative bacteria.

4. Preparation
• Mordant: These are chemical agents which have
power of making dyes to stain materials which
otherwise are unstainable
• Mordants are classified into two categories:
• a) Basic Mordant: React with acidic dyes e.g. alum ,
ferrous sulfate , cetylpyridinium chloride etc .
• b) Acidic Mordant : React with basic dyes e.g. picric
acid , tannic acid etc.
• Direct Staining: Carried out without mordant.
• Indirect Staining: Staining brought by the aid of a
mordant

5. Negative staining
• A simple staining method for bacteria that is usually
successful, even when the "positive staining" methods
detailed below fail, is to use a negative stain.
• This can be achieved by smearing the sample onto the
slide and then applying nigrosin (a black synthetic dye)
or India ink (an aqueous suspension of carbon
particles).
• After drying, the microorganisms may be viewed in
bright field microscopy as lighter inclusions well-
contrasted against the dark environment surrounding
them.
• Note: negative staining is a mild technique that may not
destroy the microorganisms, and is therefore unsuitable
for studying pathogens

6. Gram stain
• Gram stain or Gram staining, also called Gram's
method, is a method of staining used to distinguish
and classify bacterial species into two large groups
(gram-positive and gram-negative).
• The name comes from the
Danish bacteriologist Hans Christian Gram, who
developed the technique.
• Gram staining differentiates bacteria by the
chemical and physical properties of their cell walls.
Gram-positive cells have a thick layer
of peptidoglycan in the cell wall that retains the
primary stain, crystal violet.

7. • Gram-negative cells have a thinner peptidoglycan layer
that allows the crystal violet to wash out on addition of
ethanol.
• They are stained pink or red by the counterstain,
commonly safranin or fuchsine. Lugol's iodine solution
is always added after addition of crystal violet to
strengthen the bonds of the stain with the cell
membrane.
• The Gram staining is almost always the first step in the
preliminary identification of a bacterial organism.
While
• Gram staining is a valuable diagnostic tool in both
clinical and research settings, not all bacteria can be
definitively classified by this technique. This gives rise
to gram-variable and gram-indeterminate groups.

8. Summary of Gram stain
Application
of
Reagent
Cell color
Gram-positive Gram-negative
Primary dye crystal violet purple purple
Trapping
agent
iodine purple purple
Decolorizer alcohol/acetone purple colorless
Counter stain
safranin/carbol
fuchsin
purple pink

9. Principle of Gram Staining
• When the bacteria is stained with primary stain Crystal Violet and
fixed by the mordant, some of the bacteria are able to retain the
primary stain and some are decolorized by alcohol.
• The cell walls of gram positive bacteria have a thick layer of
protein-sugar complexes called peptidoglycan and lipid content is
low.
• Decolorizing the cell causes this thick cell wall to dehydrate and
shrink, which closes the pores in the cell wall and prevents the
stain from exiting the cell. So the ethanol cannot remove the
Crystal Violet-Iodine complex that is bound to the thick layer of
peptidoglycan of gram positive bacteria and appears blue or purple
in colour.
• In case of gram negative bacteria, cell wall also takes up the CV-
Iodine complex but due to the thin layer of peptidoglycan and thick
outer layer which is formed of lipids, CV-Iodine complex gets
washed off. When they are exposed to alcohol, decolorizer
dissolves the lipids in the cell walls, which allows the crystal
violet-iodine complex to leach out of the cells. Then when again
stained with safranin, they take the stain and appears red in color.

10. Equipment
• Bunsen burner, alcohol-cleaned microscope slide,
water
• Reagents
• Crystal violet, Gram's iodine solution,
acetone/ethanol (50:50 v:v), 0.1% basic fuchsin
solution

11. Procedures
• Prepare a Slide Smear:
• A. Transfer a drop of the suspended culture to be
examined on a slide with an inoculation loop. If the
culture is to be taken from a Petri dish or a slant
culture tube, first add a drop or a few loopful of
water on the slide and aseptically transfer a minute
amount of a colony from the Petri dish. Note that
only a very small amount of culture is needed; a
visual detection of the culture on an inoculation loop
already indicates that too much is taken.

12. • If staining a clinical specimen, smear a very thin layer onto
the slide, using a wooden stick. Do not use a cotton swab, if at
all possible, as the cotton fibers may appear as artefacts. The
smear should be thin enough to dry completely within a few
seconds. Stain does not penetrate thickly applied specimens,
making interpretation very difficult.
• B. Spread the culture with an inoculation loop to an even thin
film over a circle of 1.5 cm in diameter, approximately the
size of a dime. Thus, a typical slide can simultaneously
accommodate 3 to 4 small smears if more than one culture is
to be examined.
• C. Air-dry the culture and fix it or over a gentle flame, while
moving the slide in a circular fashion to avoid localized
overheating. The applied heat helps the cell adhesion on the
glass slide to make possible the subsequent rinsing of the
smear with water without a significant loss of the culture.
Heat can also be applied to facilitate drying the smear.
However, ring patterns can form if heating is not uniform, e.g.
taking the slide in and out of the flame.

13. Gram Staining
• A. Add crystal violet stain over the fixed culture. Let
stand for 10 to 60 seconds; for thinly prepared
slides, it is usually acceptable to pour the stain on
and off immediately. Pour off the stain and gently
rinse the excess stain with a stream of water from a
faucet or a plastic water bottle. Note that the
objective of this step is to wash off the stain, not the
fixed culture.
• B. Add the iodine solution on the smear, enough to
cover the fixed culture. Let stand for 10 to 60
seconds. Pour off the iodine solution and rinse the
slide with running water. Shake off the excess water
from the surface.

14. • C. Add a few drops of decolorizer so the solution
trickles down the slide. Rinse it off with water after
5 seconds. The exact time to stop is when the
solvent is no longer colored as it flows over the
slide. Further delay will cause excess decolorization
in the gram-positive cells, and the purpose of
staining will be defeated.
• D. Counterstain with basic fuchsin solution for 40 to
60 seconds. Wash off the solution with water. Blot
with bibulous paper to remove the excess water.
Alternatively, the slide may shaken to remove most
of the water and air-dried.

15. Quality control
• It is a simple matter to prepare a control slide by
breadking a clean wooden applicator stick and
picking a small amount of material from the
interproximal space of one's teeth. This should be
smeared into a drop of clean tap water on a clean
glass slide. The slide may be stained as above. This
material will consistently display a few neutrophils
and a mixture of Gram (+) and (-) organisms.
Neutrophil nuclei should be pink.

16. Examine the finished slide under a
microscope
• A caveat in the examination of the Gram smears is the
distortion in morphology that can be caused by
antimicrobial therapy. This is especially likely to occur
in urine speciments.
• Filamentous and pleomorphic forms may be observed
among the Gram (-) rod species.
• Gram reaction of the organism may also change after
antimicrobial therapy, Gram (+) bacterial may become
gram variable. Look at areas that are one cell thick
only; observation of thick areas will give variable and
often incorrect results. White blood cells and
macrophages should stain Gram-negative, whereas
sqamous epithelial cells are Gram-positive.

20. Ziehl-Neelsen staining( Acid-fast stain):
• Ziehl-Neelsen staining is a type of Acid-fast stain, first
introduced by Paul Ehrlich.
• Ziehl–Neelsen staining is a bacteriological stain used to
identify acid-fast organisms, mainly Mycobacteria. The genus
Mycobacterium is a slow growing bacteria, made up of small rods
that are slightly curved or straight, and are considered to be gram
positive.
• Some types of Mycobacteria form branches or filaments. Some
mycobacteria are free-living saprophytes, but many are pathogens
that cause disease in animals and humans.
• Mycobacterium bovis causes tuberculosis in cattle. Since
tuberculosis can be spread to humans, milk is pasteurized to kill
any of the bacteria.
• Some Mycobacteria species that cause disease in humans
include Mycobacterium leprae, Mycobacterium
kansasii, Mycobacterium marinum, Mycobacterium
bovis, Mycobacterium africanum and members of
the Mycobacterium avium complex.

21. • Mycobacterium tuberculosis is a species of Mycobacterium
that causes tuberculosis (TB). Mycobacterium tuberculosis is
an airborne bacterium that typically infects the human lungs.
• Symptoms of TB include a bad cough, chest pain, fatigue,
weight loss, no appetite, chills, fever and night sweats. The
typical regimen for treating a Latent TB infection includes the
use of isoniazid, rifapentine, and rifampin. The regimen is
changed for those who have developed a drug resistant strain
of TB.
• Testing for TB includes blood testing, skin tests, and chest x-
rays. When looking at the smears for TB, it is stained using
and acid-fast stain.
• These Acid-fast organisms like Mycobacterium contain large
amounts of lipid substances within their cell walls called
mycolic acids. These acids resist staining by ordinary methods
such as a Gram stain. It can also be used to stain a few other
bacteria, such as Nocardia. The reagents used for Ziehl–
Neelsen staining are – carbol fuchsin, acid alcohol,
and methylene blue. Acid-fast bacilli are bright red after
staining.

22. Summary of acid-fast stain (Ziehl–Neelsen stain)
Application
of
Reagent
Cell colour
Acid fast Non-acid fast
Primary dye Carbol fuchsin Red Red
Decolorizer Acid alcohol Red Colorless
Counter stain
Methylene
blue/malachite
green
Red Blue

23. Principle of Acid-Fast Stain
• When the smear is stained with carbol fuchsin, it solubilizes
the lipoidal material present in the Mycobacterial cell wall but
by the application of heat, carbol fuchsin further penetrates
through lipoidal wall and enters into cytoplasm.
• Then after all cell appears red. Then the smear is decolorized
with decolorizing agent (3% HCL in 95% alcohol) but the acid
fast cells are resistant due to the presence of large amount of
lipoidal material in their cell wall which prevents the
penetration of decolorizing solution.
• The non-acid fast organism lack the lipoidal material in their
cell wall due to which they are easily decolorized, leaving the
cells colorless.
• Then the smear is stained with counterstain, methylene blue.
Only decolorized cells absorb the counter stain and take its
color and appear blue while acid-fast cells retain the red color.

24. Procedure of Acid-Fast Stain
• Prepare bacterial smear on clean and grease free slide, using
sterile technique.
• Allow smear to air dry and then heat fix.
Alcohol-fixation: This is recommended when the smear has
not been prepared from sodium hypochlorite (bleach)
treated sputum and will not be stained
immediately. M. tuberculosis is killed by bleach and during
the staining process. Heat-fixation of untreated sputum will
not kill M. tuberculosis whereas alcohol-fixation
is bactericidal.
• Cover the smear with carbol fuchsin stain.
• Heat the stain until vapour just begins to rise (i.e. about
60ºC). Do not overheat. Allow the heated stain to remain on

25. • Heating the stain: Great care must be taken
when heating the carbol fuchsin especially if
staining is carried out over a tray or other container
in which highly fiammable chemicals have collected
from previous staining.
• Only a small fiame should be applied under the
slides using an ignited swab previously dampened
with a few drops of acid alcohol or 70% v/v ethanol
or methanol.
• Do not use a large ethanol soaked swab because this
is a fire risk.

26. • Wash off the stain with clean water.
Note: When the tap water is not clean, wash the smear
with filtered water or clean boiled rainwater.
• Cover the smear with 3% v/v acid alcohol for 5 minutes
or until the smear is sufficiently decolorized, i.e. pale
pink.
Caution: Acid alcohol is fiammable, therefore use it
with care well away from an open fiame.
• Wash well with clean water.
• Cover the smear with malachite green stain for 1–2
minutes, using the longer time when the smear is thin.
• Wash off the stain with clean water.
• Wipe the back of the slide clean, and place it in a
draining rack for the smear to air-dry (do not blot dry).
• Examine the smear microscopically, using the 100 X oil
immersion objective.

29. Albert’s staining
• Albert stain is a type of differential stain used for
staining the volutin granules also known as
Metachromatic granules or food granules found
in Corynebacterium diphtheriae.
• It is named as metachromatic because of its property
of changing colour i.e when stained with blue stain
they appear red in colour.
• When grown in Loffler’s slopes, C.
diphtheriae produces large number of granules

30. Principle of Albert Staining
• Albert stain is basically made up of two stains that is
Toluidine blue’ O’ and Malachite green both of which
are basic dyes with high affinity for acidic tissue
components like cytoplasm. The pH of
• Albert stain is adjusted to 2.8 by using acetic acid which
becomes basic for volutin granules as pH of volutin
Granule is highly acidic.
• Therefore on applying Albert’s stain to the smear,
Toluidine blue’ O’ stains Volutin Granules i. e the most
acidic part of cell and Malachite green stains the
cytoplasm blue-green.
• On adding Albert’s iodine due to effect of iodine, the
metachromatic property is not observed and granules
appear blue in colour.

31. Composition of Albert stain: Albert
stain is composed of two reagents:
• Albert’s A solution consist of
• Toludine blue 0.15 gm
• Malachite green 0.20 gm
• Glacial acetic acid 1 ml
• Alcohol (95% ethanol) 2ml
• Dissolve the dyes in alcohol and add to the distilled
water and acetic acid.
• Allow the stain to stand for one day and then filter.
• Add Distilled water to make the final volume 100ml

32. Albert’s B solution consist of
• Iodine 2gm
• Potassium iodide (KI) 3 gm
• Dissolve KI in water and then add iodine.
Dissolve iodine in potassium iodide solution
• Requirements: Smear on glass slide, staining
rack, Albert’s A solution , Albert’s B solution,
blotting paper, immersion oil, microscope.

34. Procedure
• Prepare a smear on clean grease free slide.
• Air dry and heat fix the smear.
• Treat the smear with Albert’s stain and allow it to
react for about 7 mins.
• Drain of the excess stain do not water wash the slide
with water.
• Flood the smear with Albert’s iodine for 2 minutes.
• Wash the slide with water, air dry and observe under
oil immersion lens.

35. Result
• If Corynebacterium diphtheria is present in the
sample it appears green coloured rod shaped bacteria
arranged at angle to each other, resembling English
letter ‘L’, ‘V’ or Chinese letter pattern along with
bluish black metachromatic granules at the poles.

36. Bacterial resistance to antibacterial therapy
• Antimicrobial resistance (AMR or AR) is the ability
of a microbe to resist the effects of medication that once
could successfully treat the microbe. The
term antibiotic resistance (AR or ABR) is a subset of
AMR, as it applies only to bacteria becoming resistant
to antibiotics.
• Resistant microbes are more difficult to treat, requiring
alternative medications or higher doses of
antimicrobials. These approaches may be more
expensive, more toxic or both.
• Microbes resistant to multiple antimicrobials are
called multidrug resistant (MDR). Resistance arises
through one of three mechanisms: natural resistance in
certain types of bacteria, genetic mutation, or by one
species acquiring resistance from another. All classes of
microbes can develop resistance.

37. • Fungi develop antifungal resistance.
• Viruses develop antiviral resistance. Protozoa develop a
ntiprotozoal resistance,
and bacteria develop antibiotic resistance. Resistance
can appear spontaneously because of random mutations.
• However, extended use of antimicrobials appears to
encourage selection for mutations which can render
antimicrobials ineffective.
• Preventive measures include only using antibiotics
when needed, thereby stopping misuse of antibiotics or
antimicrobials.
• Narrow-spectrum antibiotics are preferred over broad-
spectrum antibiotics when possible, as effectively and
accurately targeting specific organisms is less likely to
cause resistance, as well as side effects.

38. • For people who take these medications at home,
education about proper use is essential.
• Health care providers can minimize spread of resistant
infections by use of proper sanitation and hygiene,
including handwashing and disinfecting between
patients, and should encourage the same of the patient,
visitors, and family members.
• Rising drug resistance is caused mainly by use of
antimicrobials in humans and other animals, and spread
of resistant strains between the two.
• Growing resistance has also been linked to dumping of
inadequately treated effluents from the pharmaceutical
industry, especially in countries where bulk drugs are
manufactured.

39. • Antibiotics increase selective pressure in bacterial
populations, causing vulnerable bacteria to die; this increases
the percentage of resistant bacteria which continue growing.
• Even at very low levels of antibiotic, resistant bacteria can
have a growth advantage and grow faster than vulnerable
bacteria. With resistance to antibiotics becoming more
common there is greater need for alternative treatments.
• Calls for new antibiotic therapies have been issued, but new
drug development is becoming rarer.
• Antimicrobial resistance is increasing globally because of
greater access to antibiotic drugs in developing countries.
• Estimates are that 700,000 to several million deaths result per
year. Each year in the United States, at least 2.8 million people
become infected with bacteria that are resistant to antibiotics
and at least 35,000 people die as a result.

40. • There are public calls for global collective action to
address the threat that include proposals
for international treaties on antimicrobial resistance.
• Worldwide antibiotic resistance is not completely
identified, but poorer countries with weaker
healthcare systems are more affected.
• The WHO defines antimicrobial resistance as a
microorganism's resistance to an antimicrobial drug
that was once able to treat an infection by that
microorganism.
• A person cannot become resistant to antibiotics.
Resistance is a property of the microbe, not a person
or other organism infected by a microbe.

41. • Antibiotic resistance is a subset of antimicrobial resistance.
• This more specified resistance is linked to pathogenic bacteria
and thus broken down into two further subsets,
microbiological and clinical. Resistance linked
microbiologically is the most common and occurs from genes,
mutated or inherited, that allow the bacteria to resist the
mechanism associated with certain antibiotics.
• Clinical resistance is shown through the failure of many
therapeutic techniques where the bacteria that are normally
susceptible to a treatment become resistant after surviving the
outcome of the treatment. In both cases of acquired resistance,
the bacteria can pass the genetic catalyst for resistance
through conjugation, transduction, or transformation.
• This allows the resistance to spread across the same pathogen
or even similar bacterial pathogens.

42. Causes
• Bacteria with resistance to antibiotics predate medical
use of antibiotics by humans. However, widespread
antibiotic use has made more bacteria resistant through
the process of evolutionary pressure.
• Reasons for the widespread use of antibiotics in human
medicine include:
• increasing global availability over time since the 1950s
• uncontrolled sale in many low or middle income
countries, where they can be obtained over the counter
without a prescription, potentially resulting in
antibiotics being used when not indicated. This may
result in emergence of resistance in any remaining
bacteria.

43. Other causes include
• Antibiotic use in livestock feed at low doses for
growth promotion is an accepted practice in many
industrialized countries and is known to lead to
increased levels of resistance.
• Releasing large quantities of antibiotics into the
environment during pharmaceutical manufacturing
through inadequate wastewater treatment increases
the risk that antibiotic-resistant strains will develop
and spread.
• It is uncertain whether antibacterials in soaps and
other products contribute to antibiotic resistance,
but antibacterial soaps are discouraged for other
reasons.

44. Antiseptics create AMR to antibiotics and other antiseptics
• Antiseptics appear to activate tolerance mechanisms in
bacteria, which offer them protection against a range of
antiseptics as well as antibiotics. Antiseptics are used for
cleaning in hospitals and in many wound care dressings.
These findings may explain the increase in treatment-resistant
hospital infections.
• Exposure to low doses of the antiseptic octenidine allowed
several different strains of Pseudomonas aeruginosa to
develop cross-tolerance to other antiseptics and to several
different antibiotics.
• The level of tolerance was substantial, i.e. in several cases a
32-fold increase in concentrations of the antiseptic was
required to obtain the same antimicrobial effect. Also, this
increased resistance was permanent.
• The same group also reported that Klebsiella pneumoniae was
able to develop tolerance to chlorhexidine and that 5 out of 6
strains showed cross-resistance to the last-resort antibiotic,
colistin.

45. Clinical significance
• Increasing bacterial resistance is linked with the volume of
antibiotic prescribed, as well as missing doses when taking
antibiotics. Inappropriate prescribing of antibiotics has been
attributed to a number of causes, such as patients insisting on
antibiotics and physicians prescribing them as they do not have
time to explain why they are not necessary.
• Another cause can be physicians not knowing when to prescribe
antibiotics or being overly cautious for medical or legal reasons.
• For example, 70 to 80 percent of diarrhea is caused by viral
pathogens, for which antibiotics are not effective. But nevertheless,
around 40 percent of these cases are attempted to be treated with
antibiotics.
• In some areas even over 80 percent of such cases are attempted to
be treated with antibiotics.
• Also, hospitals are often unable to identify the causative
organism(s) in time to treat patients presenting with rapidly
progressing sepsis or other severe infections, resulting in the
overuse of broad-spectrum antibiotics.

46. Prevention
• There have been increasing public calls for global
collective action to address the threat, including a
proposal for international treaty on antimicrobial
resistance.
• Further detail and attention is still needed in order to
recognize and measure trends in resistance on the
international level; the idea of a global tracking system
has been suggested but implementation has yet to occur.
• A system of this nature would provide insight to areas
of high resistance as well as information necessary for
evaluation of programs and other changes made to fight
or reverse antibiotic resistance.

47. • Lower antibiotic concentration contributes to the
increase of AMR by introducing more mutations that
support bacterial growth in higher antibiotic
concentration. For example, sub-inhibitory
concentration have induced genetic mutation in bacteria
such as Pseudomonas aeruginosa and Bacteroides
fragilis.
• Up to half of antibiotics used in humans are
unnecessary and inappropriate. For example, a third of
people believe that antibiotics are effective for
the common cold, and the common cold is the most
common reason antibiotics are prescribed even though
antibiotics are useless against viruses.
• A single regimen of antibiotics even in compliant
individuals leads to a greater risk of resistant organisms
to that antibiotic in the person for a month to possibly a
year.

48. • Antibiotic resistance increases with duration of
treatment. Therefore, as long as an effective minimum
is kept, shorter courses of antibiotics are likely to
decrease rates of resistance, reduce cost, and have better
outcomes with fewer complications.
• Short course regimens exist for community-acquired
pneumonia spontaneous bacterial peritonitis, suspected
lung infections in intense care wards, so-called acute
abdomen, middle ear infections, sinusitis and throat
infections, and penetrating gut injuries. In some
situations a short course may not cure the infection as
well as a long course.
• A BMJ editorial recommended that antibiotics can often
be safely stopped 72 hours after symptoms resolve.

49. • Because individuals may feel better before the infection is
eradicated, doctors must provide instructions to them so they know
when it is safe to stop taking a prescription. Some researchers
advocate doctors' using a very short course of antibiotics,
reevaluating the patient after a few days, and stopping treatment if
there are no clinical signs of infection.
• Certain antibiotic classes result in resistance more than others.
Increased rates of MRSA (methicillin-resistant Staphylococcus aureus )
infections are seen when using glycopeptides, cephalosporins,
and quinolone antibiotics. Cephalosporins, and particularly
quinolones and clindamycin, are more likely to produce
colonisation with Clostridium difficile.
• Factors within the intensive care unit setting such as mechanical
ventilation and multiple underlying diseases also appear to
contribute to bacterial resistance. Poor hand hygiene by hospital
staff has been associated with the spread of resistant organisms.
• Counterfeit medications may contain sub-therapeutic
concentrations of antibiotics, designed to reduce the chance of
detection, and this by definition, increases antimicrobial resistance.