The document discusses various bacterial staining techniques essential for visualizing bacteria under a microscope, including simple staining, negative staining, differential staining (gram stain and acid-fast stain), and structural staining (spore stain and capsule stain). It explains the types of dyes used, staining procedures, and the differences between gram-positive and gram-negative bacteria in terms of their cell wall structures. Additionally, it covers the importance of these staining methods in microbiology for identifying and classifying microorganisms.

1. Bacterial staining
Made by
Sameh aly gad
Demonstrator of microbiology and immunology
College of pharmacy
Minia university

2. outlines
Dyes(stains)
Negative staining
Structural staining
Spore stain
Capsule stain
Simple staining
Differential staining
Gram stain
Acid-fast stain

3. Dyes
 Why we need to stain bacteria?
 Bacteria are transparent and colorless , so they would be
invisible to naked eye if observed under a microscope
thus
bacteria should be stained with certain dyes in order to
visualize bacterial cell or their internal structures using
the light microscope.

4. Dyes (continued)
 Dye (stain):
colored organic compound in the form of salt,
composed of positive and negative ion, one of these ions
is responsible for colour called chromogen.
 Types of Dyes:
 Basic dyes
 Acidic dyes

5. Dyes (continued)
 Basic dyes:
 In which chromogen is the positive ion (cation).
 Basic dye has the form: dye+ Cl-
 examples include crystal violet, methylene blue and safranin.
 Acidic dyes:
 In which chromogen is the negative ion (anion).
 Acidic dye has the form: Na+dye-
 Examples include nigrosin and India ink.

6. Simple staining
 Simple staining is carried out to visualize bacteria and to
compare morphological shapes and arrangements of bacterial
cells.
 In simple stain, the bacterial smear is stained with a single
basic dye.
 Bacterial cell surface is slightly negative so it tends to bind
strongly to the cationic chromogen of basic dyes.
 Thus , basic dyes are used to stain bacterial cells

7. Simple staining procedure
1. Add one loopful of the sample onto a glass slide.
2. Allow it to air-dry.
3. Heat-fix the specimen on the glass slide ,unless the specimen
is heat-fixed ,the bacterial smear will wash away during the
staining procedure.
4. Staining: crystal violet and wait for 1 min. or safranin and
wait for 3-4 min.
5. Wash the smear with tap water to remove the excess stain.
6. Blot dry, then add cedarwood oil and examine under a
microscope. Sameh aly gad

8. Bacterial shapes and arrangement

9. Bacillus subtilis stained with crystal violet Saccharomyces stained with crystal violet
Staphylococci stained with crystal violet

10. Negative staining
 It is a type of simple staining in which acidic dyes such as
India ink or nigrosin are used.
 The acidic dye with its negatively charged chromogen will not
penetrate the cell due to the repulsion with the negatively
charged bacterial surface.
 As a result the bacterial cells remain unstained but easily
discernible against the coloured background so it is also called
indirect stain.
 Since no heat is used, negative stain is used to visualize cells
that are too delicate to be heat-fixed.

12. Negatively stained staphylococci

13.  Differential staining
a) Gram stain b) Acid fast stain

14. Gram stain
 Most bacteria are classified into two main groups: gram
positive and gram negative bacteria.
 Comparison between gram positive and gram negative
bacteria.

15. Gram stain (cont…)
 It is a differential stain which distinguishes between gram
positive and gram negative bacteria depending on the
difference in the cell wall structure.
 Principle:
• Gram positive bacteria when treated with a basic dye such as
crystal violet , then with iodine (will fix the stain to the cell)
and then subjected to a subsequent treatment with a
decolourizing agent such as alcohol/acetone mixture remain
stained with the basic dye and the decolourizer doesn,t
remove the dye ,

16. Gram stain (cont…)
however, gram negative bacteria when treated similarly will be
readily decolourized with the alcohol acetone mixture and
become invisible , thus to render the unstained gram negative
bacteria visible, a counterstain is applied.
 Why gram positive bacteria resist decolourization by
alcohol/acetone mixture but the gram negative ones can not?
 Because the cell wall of gram negative bacteria has high lipid
content and low peptidoglycan content so when treated with
acetone/alcohol mixture dissolves their lipids creating pores

17. Gram stain (cont…)
In the cell wall through which crystal violet dye will diffuse,
and gram negative bacteria become unstained.
On the other hand, gram positive bacteria has high
peptidoglycan content and low lipid content so when
treated with the decolourizer, it will dehydrate the cell
wall causing it to shrink and the pores to close and thus
the diffusion of the dye will be blocked

18. Gram stain procedure
Prepare The Smear On a Glass Slide , Air Dry
Heat Fixation
Flood With Crystal Violet (primary stain)
Washing with water

19. Flood with gram‘s iodine, Washing with water
Decolorization with acetone/alcohol mixture
Washing with water
Flood with safranin(2 ry stain), washing with water
Blot dry using paper, Examine with the microscope

20. Gram stain (cont…)
 If the specimen contains both gram positive and gram
negative bacteria, gram positive ones appear violet while
gram negative ones appear pink to red
Gram stained staphylococci
Type of stain: differential stain
Name of stain: gram stain
Gram reaction: positive gram
Colour: violet
Size: small
Shape: spherical or cocci
Arrangement: grapelike clusters
Name of m.o: Staphylococcus sp.

21. Gram stained bacillus sp.
Type of stain: differential stain
Name of stain: gram stain
Gram reaction: positive gram
Colour: violet
Size: intermediate
Shape: rod-shaped
Arrangement: chains
Name of m.o: Bacillus sp.
Type of stain: differential stain
Name of stain: gram stain
Gram reaction: positive gram
Colour: violet
Size: large
Shape: oval
Arrangement: single
Gram stained saccharomyces Name of m.o: Saccharomyces sp.

22. Gram stained Escherichia coli
Type of stain: differential stain
Name of stain: gram stain
Gram reaction: gram negative
Colour: pink
Size: small
Shape: rod shaped
Arrangement: single
Name of m.o: Escherichia coli
Mixture of gram positive
staphylococci and gram
negative E.coli

23. Acid-fast stain(Ziehl-Neelsen stain)
 Members of the genus mycobacterium are resistant to simple
and gram staining procedure due to their thick waxy (fatty)
cell wall which makes the penetration of dyes extremely
difficult and requires the application of heat.
 But once the dye has penetrated, it can not be removed even
with the vigorous use of acid alcohol as decolourizing agent,
due to this property, these organisms are called acid fast.
 The most important human pathogens of them are
mycobacterium tuberculosis and mycobacterium leprae.

24. Acid-fast stain procedure
1. Prepare a smear of the microorganism on a clean slide.
2. Allow the smear to air-dry then fix it by flaming.
3. Place the slide over a steam bath and flood the smear with
strong carbol fuchsin and keep it for 5 mins. caution :don,t
allow stain to dry; replenish stain as needed. also, prevent
stain from boiling by adjusting the temperature applied.
4. Allow to cool and then wash with tap water.
5. Decolourize with acid alcohol, till the effluent runs almost
clear with a slight red tinge.

25. Acid-fast stain procedure
6. Wash with tap water
7. Counterstain with methylene blue for 2 mins.
8. Wash smear with tap water.
9. Blot dry and examine with a microscope.
Acid fast bacteria
appear red while
non acid fast
bacteria appear
blue.

26.  Structural stain
a) Spore stain b) Capsule stain
c) Flagella stain

27. Spore stain
(Schaeffer-Fulton method)
 Some gram positive bacteria especially members of the
genera clostridium and bacillus have the ability to form
spore(endospore) when exposed to drastic unfavorable
conditions such as drying, heating, lack of nutrients and so
on.
 Spores formed by these bacteria are highly resistant to
unfavorable conditions and can remain viable for several
years, once the conditions become favorable the spore
germinates.

28. Spore stain(cont..)
 Spores don,t accept the stain easily due to their thick
impervious coat and require the use of heating during
staining.
Procedure:
1. Prepare smear of the microorganism, allow to dry and the
heat-fix to a slide.
2. Place the slide over a steam bath, flood the smear with
malachite green dye and keep it for 3-5 mins, replenish
smear with malachite green dye if some evaporates and don't
allow the stain to boil.

29. Spore stain(cont..)
3. Allow the slide to cool and wash with tap water to remove
excess stain.
4. Counterstain with safranin for 2 min.
5. Rinse the slide with running tap water to remove excess stain.
6. Blot dry with bibulous paper and examine under oil
immersion.

30. Capsule stain
 It is a well organized gelatinous layer that is secreted by the
cell, surrounds and adheres to the cell wall.
 Capsule is not common to all organisms and the organisms
that have a heavy capsule are generally virulent and capable
of producing disease.
 It protects bacteria against the phagocytic activities of host
phagocytes.
 It also serves as a barrier against antimicrobials preventing
them from entering the cell.

31. Capsule stain(cont..)
 Most capsules are polysaccharides (ex: Streptococcus
pneumoniae) in nature but some are glycoproteins or
polypeptides.
 Since capsule fails to retain standard dyes, it can't be stained
by simple staining procedure or even gram stain, but it can be
visualized indirectly using negative staining technique.
 A basic dye is used to stain the bacterial cell and acidic dye is
used to colour the background where the capsule appears as a
clear halo zone between the coloured background and the
stained cell.

32. Capsule stain(cont..)
Procedure
 Prepare a smear from the encapsulated microorganism( ex.
Klebsiella pneumoniae) without heat fixation.
 Stain with safranin for 5-6 mins.
 Wash with tap water(polysaccharide capsules are water-soluble
thus gentle washing is required, otherwise the capsule
will be removed with water).
 Put a drop of nigrosin at one end of the slide and spread it
with another slide.
 Allow it to air-dry and examine under oil immersion.

33. Figure showing indirectly stained capsule (Klebsiella pneumoniae capsule)