This chapter discusses staining techniques used in hematology. It covers the principles of staining, types of stains including Giemsa, Gram, and acid-fast bacilli (AFB) stains. Specific details are provided on staining blood films, smears, the appearance of stained cells, and staining procedures and results for different techniques. The chapter aims to explain staining principles and identify different staining methods and their uses in identifying cells and microorganisms.

1. Chapter 3
Staining techniques
By: Ahmedmenewer (Msc
)
12/12/20191

2. Learning Objectives
At the end of this chapter, the student will be
able to:
 Explain the general principle of staining in blood
films in hematology
 Identify type of staining technique
 Describe the appearance of cells and cell
components stained blood films
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3. Outline
 Introduction
 Principle of staining
 Blood film(BF)
 Giemsa Stain
 Gram stain
 AFB stain
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4. Introduction
Stains (Dyes) are coloured chemical
compounds that
are used to selectively give colour to the
colourless structures of bacteria or other cells.
Bacterial staining is the process of giving
colour to the colourless structures (cell wall,
spore, etc) of the bacteria in order to make it
visible under the microscope.
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5. Uses of staining
1. To observe the morphology, size and
arrangement of cell
2. To differentiate one group of bacteria from
the other group.
Staining reactions are made possible because
of the
Physical phenomena of capillary osmosis,
solubility,
adsorption, and absorption of stains or dyes
by cells of
micro-organisms. 12/12/20195

6. Principle of Staining
 Acidic dyes such as eosin unite with the basic
components of the cell (cytoplasm)
 Conversely, basic stains like methylene blue are
attracted to and combine with the acidic parts of the
cell (nucleic acid and nucleoproteins of the nucleus.
 Other structures stained by combination of the two
are neutrophilic
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7. Steps for Blood Film

8. The shape of blood film

9. Examples of unacceptable
smears

10. Giemsa stain
 Employs various compounds (thionine and its
methyl derivative) with eosin and methylene blue
 Is an alcohol-based Romanowsky stain that
requires dilution in pH 7.1-7.2 buffered water
 It is excellent in staining malaria parasites in thick films.
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11. Indications of the different stains for
use
 Wright stain
 Peripheral smears
 Leishamn
 Peripheral smears
 Geimsa
 For malaria thick films
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12.  Cytoplasm
 Monocytes – gray blue with fine reddish granules
 Neutrophils: light pink with lilac (pale purple) granules
 Lymphocytes: varying shades of blue
 Malaria parasites – sky blue cytoplasm and red purple
chromatin
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Appearance of cells and cell components in -stained
blood films

15. Increased neutrophils count (neutrophilia)
1. Acute bacterial infection.
2. Many inflammatory processes.
3. During physical stress.
4. With tissue necrosis.
5. Granulocytic leukemia.
Decreased neutrophil count (neutropenia)
1. Typhoid fever
2. Brucellosis
3. Viral diseases, including hepatitis, influenza,
rubella, and mumps.
4. A great infection can also deplete the bone
marrow of neutrophils.
5. Many drugs used to treat cancer produce bone
6. marrow depression.

16. Bacteriological staining
 Why we need to make smears?
 Making smear is a precondition to facilitate
staining and further observation of
microorganisms under microscope.
 Without making appropriate smear, which is thin
enough to make a single layer of bacteria, it is
difficult to observe and read different staining
reactions of bacteria. .
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17. Preparing smear
 If smears are to provide reliable information they must be
prepared, labeled and fixed correctly prior to being
stained.
Labeling Slides
 Every slide must be labeled clearly with the date and the
patients name and number. When ever possible, smears
should be spread on slides which have one end frosted
for labeling
 A lead pencil should be used for writing on the
frosted area. Because pencil marks unlike grease
pencil marks, will not be washed off during the
staining processes.
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18. The thickness of the smear should allow to read a text when
placed under the smear.
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19. PRINCIPLE OF STAINING IN
BACTERIOLOGY
 Even with the microscope, bacteria are difficult to see
unless they are treated in a way that increases contrast
between the organisms and their background.
 The most common method to increase contrast is to
stain part or all of the microbe.
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20.  The cellular components of mammalian as well as
microbial cell are different.
 For example the nuclei of cell is negatively charged
because of the presence of acidic component (DNA)
hence it combines with positively charged compounds,
(basic dyes). and the cytoplasm parts of a cell is
generally positively charged therefore combines with
negatively charged compounds (acidic dyes).
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PRINCIPLE OF STAINING IN
BACTERIOLOGY

21. GRAM’S STAIN
 This method was developed by the Danish
bacteriologist
Christian Gram in 1984.
Basic concepts:
 Most bacteria are differentiated by their gram reaction
due to differences in their cell wall structure.
 The surface of bacterial cell has got a negative
charge due to the presence of polysaccharides and
lipids (PG) this has made the surface of the bacteria
to have affinity to cationic or basic dyes (when the
colouring part is contained in the basic part.)
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22. Principle of Gram’s stain
 The principle of Gram’s stain is that cells are first fixed to
slide by heat or alcohol and stained with a basic dye (e.g.
crystal violate), which is taken up in similar amounts by all
bacteria.
The slides are then treated with an Gram’s iodine
(iodine KI mixture) to fix (mordant) the crystal violet stain
on Gram positive bacteria, decolorized with acetone or
alcohol, and finally counter stained with Safranin.
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23. Gram positive bacteria
 Gram positive bacteria: - stain dark purple with
crystal violet and are not decolorized by acetone or
ethanol. The following are some important examples
of gram positive bacteria.
 Staphylococcus,
 Streptococcus,
 Clostridium
 Bacillus
 Corynebacterium …
 N.B. The reason for the retention of the primary
stain (CV) by the gram positive bacteria after
decolorization is due to the presence of more acidic
protoplasm (PG layer) of these organisms which
bind to the basic dye.
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24. Gram negative bacteria
Gram negative bacteria: - stain red because
after being stained with crystal violet they are
decolorized by acetone or ethanol and take up
red counter stain. (Neutral red, Safranin or
dilute carbol fuchsin).
 The following are some important gram
negative bacteria:-
 Nesseria spp.
 Haemophilus spp.
 Salmonella, shigella, vibrio, Klebsilla, Coliforms
…etc.
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25. Reagents for Gram stain
Required reagents
 Crystal violet
 Gram’s Iodine
 Acetone-Alcohol or 95% Alcohol
 Safranin or Neutral red
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26. Results of Gram’s stained smear
Results
 Gram positive bacteria …..………………….. Purple(blue)
 Yeast cells ……………………………………. Dark purple
 Gram negative bacteria …….……………….. Pale to red
 Nuclei of pus cell …….………….…………… Red
 Epithelia cells …………………………………. Pale red
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27. Figure: Gram positive and Gram negative bacteria
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28. Report of Gram’s stained smear
1. Number of bacteria present whether many, moderate, few or
scanty
2. Gram reaction of the bacteria whether Gram positive or
Gram negative
3. Morphology and arrangement of the bacteria whether cocci,
diplococci, streptococci, rods, or coccobacili; also whether
the organisms are intracellular.
4. Presence and number of pus cells.
5. Presence of yeast cells and epithelia cells.
Example of a gram stain report 12/12/201928

29. Ziehl-Neelson (Acid fast Bacilli-AFB) staining
method
 Ziehl-Neelson stain is used for staining mycobacteria
which are hardly stained by Gram‘s staining method.
 Once the Mycobacteria is stained with primary stain it
can not be decolorized with acid, so named as acid-fast
bacteria.
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30. - Mycobacteria typically are slightly bent or curved slender
rods. About 2um- 4 um long and 0.2 um – 0.5 um wide.
- The most striking chemical feature of mycobacteria is their
extra ordinary high lipid content in the cell wall (up to 60%
of its dry weight). This high lipid content probably accounts
for some of the other unusual properties of mycobacteria.
E.g Relative impermeability to stains, acid fastness, unusual
resistance to killing by acid and alkali.
 The cell wall of Mycobacteria also contains a peptido-
glycan layer, glycolipids, protein and Mycolic acid(This is
unique to mycobacteria, nocardiae and corynebacteria).
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Ziehl- Neelson (Acid fast Bacilli-AFB) staining
method

31. Principle of Ziehl-Neelson (Acid fast) staining method
 Sputum smear is heat –fixed, flooded with a solution of
carbilfusin (a mixture of basic fuschin and phenol) and
heated until steam rises. The heating which facilitate
penetration (entrance) of the primary stain into the
bacterium.
 After washing with water, the slide is covered with 3% HCl
(decolourizer). Then washed with water and flooded with
methylene blue ( Mycobacterium tuberculosis) and
malachite green (Mycobacterium leprae).
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32. Photogenic Mycobacteria
1. Tubercle bacilli
- M. tuberculosis (human tubercle type)
- M. bovis (bovine type)
2. Leprosy bacilli
- M. leprae
- M. lepraemurium
3. Environmental mycobacteria (atypical, anonymous)
mycobacteria
- M. avium- intercellulare
- M. xenopi etc
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33. Materials for AFB staining
 Sputum container (for M. tuberculosis) – for sample collection
 Wire loop or applicator stick – to spread sputum on the microscope
slide
 Microscope slide – for making smears
 Marking pen – to put identification number on the microscope slide
 Forceps- to hold smeared slide
 Bunsen burner or sprit lamp – to fix the smeared slide and to flame
the smear during staining.
 Staining racks (staining rods) – for staining.
 Slide rack – to place stained slide to dry in the air
 Ziehel – Neelson (AFB) stain - Carbolfuchsin
- 3% Acid alcohol
- Methylene blue (malachite green)
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34. M. tuberculosis
 Most infections with M. tuberculosis are caused by
inhaling cough droplets or dust particles containing
tubercle bacilli which become lodged in the lung
forming a small inflammatory lesion and cause
Pulmonary tuberculosis.
 Infected droplets remain air born for considerable
time, and may be inhaled by susceptible persons
 Tuberculosis is a disease of global importance. One
third of the world’s population is estimated to have
been infected with M. tuberculosis and eight million
new cases of tuberculosis arise each year.
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35. M. bovis
 Is found mainly as a pathogen in a cattle and
occasionally in other animals.
 Humans become infected by close contact
with infected cattle or by ingesting the
organisms in raw un treated milk.
 Person to person transmission of bovine
strains may also occur.
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36. M. Leprae
 causes leprosy, a chronic infectious disease that affects
the skin, peripheral nerves, mucosa of the upper
respiratory tact and the eye.
 It is mainly transmitted via the respiratory tract or skin
and has a long period of incubation(2 – 5 years) or
latency
Environmental Mycobacteria –
 are the most frequent causes of pulmonary infections resembling
tuberculosis, and disseminated disease and also cause lymphadenitis
(mainly in children).
 Such species are acid fast but differ from the M. tuberculosis complex
by being opportunistic pathogens, with limited distribution and
acquired from the environment. E.g Soil or water.
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37. Other forms of tuberculosis
 Tuberculous meningitis – when tubercle bacilli
reach the meninges through blood and affect
the meninge.
 Miliary tuberculosis: - Wide spread miliary
infection (liver, spleen & lymph glands)
 Renal and urogental tuberculosis
 Bone and joint tuberculosis
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38. Results of AFB STAIN
 AFB............. Red, straight or slightly curved rods,
occurring single or in a small groups
 Cells......................................... Blue
 Background Material ……….. Blue
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Fig. AFB under the microscope

39. Report of Sputum Smear
 When any definite red bacilli are seen report the
smear as AFB positive and give an indication of the
number of bacteria present as follows:
When
1- 9 AFB /100 fields --------------- report the exact
number
10 – 100 AFB/100 fields ------------------ report +
1 – 10 AFB/field --------------------------- report ++
More than 10 AFB/field ------------------ report +++
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40. 12/12/201940
Thank you