Staning in microbiology Dr Ahmed Abonar Faculty of midicine Alazhar university

2. Staining in Microbiology
By:
Dr/ Ahmed Abonar
M.Sc. Clinical Pathology

3. • Bacteria are microscopic organisms. They
are also colorless for the most part. In order
to visualize them to study their structure,
shape and other structural characteristics, it
becomes necessary to make them more
easily visible.
• This means that the structures have to be
contrasted from their environment so that
they can be seen easily.

4. • The purpose of staining is to increase the
contrast between the organisms and the
background so that they are more readily seen
in the light microscope.

5. • Simple staining: A stain which provides color
contrast but gives same color to all bacteria.
Ex: Loeffler’s methylene blue, Diluted carbol
fuchsin.
• Differential staining: A stain which imparts
different colors to different bacteria(which
contains more than one stain).
EX: Gram’s stain, Acid fast stain, Special stains.

6. How to prepare and fix smears prior
to staining
• If smears are to provide reliable information
they must be prepared, labelled, and fixed
correctly prior to being stained.
• Every slide must be labelled clearly with the
date and the patient’s name and number.

7. How to make smears
• Smears should be spread evenly covering an
area of about 15–20 mm diameter on a slide.
• The techniques used to make smears from
different specimens are as follows:

8. Purulent specimen: Using a sterile wire loop,
make a thin preparation. Do not centrifuge a
purulent fluid, e.g. c.s.f. containing pus cells.
 Non-purulent fluid specimen: Centrifuge the
fluid and make a smear from a drop of the
well-mixed sediment, e.g. direct smear from
urine or other biological fluids.

9.  Sputum for gram stain: Use a piece of clean
stick to transfer and spread purulent and
caseous material on a slide.
Culture: Emulsify a colony in sterile distilled
water and make a thin preparation on a slide.
When a broth culture, transfer a loopful to a
slide and make a thin preparation.

10.  Swabs: Roll the swab on a slide. This is
particularly important when looking for
intracellular bacteria such as N. gonorrhoeae
(urethral, cervical, or eye swab). Rolling the
swab avoids damaging the pus cells.
Faeces: Use a piece of clean stick to transfer
pus and mucus to a slide.

13. Drying and fixing smears
• After making a smear, leave the slide in a safe
place for the smear to air-dry.
• The purpose of fixation is to preserve
microorganisms and to prevent smears being
washed from slides during staining.

14. • Smears are fixed by heat, alcohol, or
occasionally by other chemicals.
• Heat fixation: This is widely used but can
damage organisms and alter their staining
reactions especially when excessive heat is
used.

15. • When used, heat fixation must be carried out
with care. The following technique is
recommended:
o 1- Allow the smear to air-dry completely.
o 2- Rapidly pass the slide, smear uppermost,
three times through the flame of a spirit lamp
or pilot flame of a Bunsen burner.
o 3- Allow the smear to cool before staining it.

16. • Heat fixation also damages leucocytes and is
therefore unsuitable for fixing smears which
may contain intracellular organisms such as N.
gonorrhoeae and N. meningitidis.

17. • Alcohol fixation: This form of fixation is far less
damaging to microorganisms than heat. Cells,
especially pus cells, are also well preserved.
• Alcohol fixation is therefore recommended for
fixing smears when looking for Gram negative
intracellular diplococc.

18. Precautions to take when staining
smears
1. Use a staining rack. Do not immerse slides in
containers of stain because this can lead to
contamination of stains and transfer of organisms
from one smear to another.
2. Do not attempt to stain a smear that is too thick.
This is one of the commonest causes of poor
staining and incorrect reporting of smears.

19. 3. Label clearly stains and reagents.
4. When washing smears of CSF sediment and
other specimens which can be easily washed
from a slide, direct the water from a wash
bottle on the back of the slide, not directly
on the smear.

20. 5. After staining, place the slides at an angle in
a draining rack for the smears to air-dry.
6. To check staining results, use quality control
smears of organisms, particularly when a
new batch of stain is used.

21. Gram stain technique
• The Gram staining reaction is used to help identify
pathogens in specimens and cultures by their Gram
reaction (Gram positive or Gram negative) and
morphology.
1. Crystal violet stain.
2. Lugol’s iodine Reagent.
3. Acetone–alcohol decolorizer.
4. Safranin (counter stain).

22. Gram positive bacteria
• Stain dark purple with crystal violet and are
not decolorized by acetone or ethanol.
Examples include species of: Staphylococcus,
Streptococcus.

23. Gram negative bacteria
• Stain red because after being stained with
crystal violet they are decolorized by acetone
or ethanol and take up the red counterstain.
Examples include species of: Neisseria,
Klebsiella, Salmonella, Shigella.

26. Steps of gram stain
1. Cover the fixed smear with crystal violet stain
for 30–60 seconds.
2. Rapidly wash off the stain with clean water.
Note: When the tap water is not clean, use
filtered water or clean boiled rainwater.
3. Tip off all the water, and cover the smear with
Lugol’s iodine for 30–60 seconds.
4. Wash off the iodine with clean water.
5. Decolorize rapidly (few seconds) with acetone–
alcohol. Wash immediately with clean water.

27. 6. Cover the smear with Safranin (counter stain)
for 2 minutes.
7. Wash off the stain with clean water.
8. Wipe the back of the slide clean, and place it
in a draining rack for the smear to air-dry.
9. Examine the smear microscopically with the
oil immersion objective to report the bacteria
and cells.

29. Interpretation of gram stains
Results:
• Gram positive bacteria . . . . . . . . . . . . Dark purple
• Yeast cells (Candida) . . . . . . . . . . . . . Dark purple
• Gram negative bacteria . . . . . . . . Pale to dark red
• Nuclei of pus cells . . . . . . . . . . . . . . . . . . . . . . Red
• Epithelial cells . . . . . . . . . . . . . . . . . . . . . . Pale red

30. Reporting gram smears:
The report should include the following information:
 Numbers of bacteria present, whether many,
moderate, few, or scanty.
 Gram reaction of the bacteria, whether Gram
positive or Gram negative.
 Morphology of the bacteria, whether cocci,
diplococci, streptococci, rods, or coccobacilli.
 Presence and number of pus cells.
 Presence of yeast cells and epithelial cells.

38. Variations in Gram reactions
 Gram positive organisms may lose their
ability to retain crystal violet and stain
Gram negatively for the following
reasons:
 Cell wall damage due to antibiotic
therapy or excessive heat-fixation of the
smear.
 Over-decolorization of the smear.

39.  Use of an iodine solution which is too old, i.e.
yellow instead of brown in colour (always
store in a brown glass or other light opaque
container).
Smear has been prepared from an old culture.
 Gram negative organisms may not be
fully decolorized and appear as Gram
positive when a smear is too thick.

40. Control of gram stain
Always check new batches of stain and
reagents for correct staining reactions using a
smear containing known Gram positive and
Gram negative organisms.

41. Ziehl Neelsen Staining
Principle, Procedure and
Interpretations

42. • The Ziehl-Neelsen (Zn) technique is used to
stain Mycobacterium species including M.
tuberculosis, M.ulcerans, and M. leprae.
• Mycobacteria, unlike most other bacteria, do
not stain well by the Gram technique.
• They can however be stained with carbol
fuchsin combined with phenol. The stain binds
to the mycolic acid in the mycobacterial cell
wall.

43. • After staining, an acid decolorizing
solution is applied. This removes the red
dye from the background cells, tissue
fibres, and any organisms in the smear
except mycobacteria which retain (hold
fast to) the dye and are therefore
referred to as acid fast bacilli, or simply
AFB.

44. • Following decolorization, the smear is
counter stained with methylene blue
which stains the background material,
providing a contrast color against which
the red AFB can be seen.

46. Preparation and fixation of the smear for
the detection of M. tuberculosis
 Sputum for Z.N. stain:
The specimen must be sputum, not saliva.
Sputum is best collected in the morning soon
after the patient wakes and before any mouth-
wash is used.
 When pulmonary tuberculosis is suspected,
up to three specimens may need to be
examined to detect AFB.

47.  The chances of detecting AFB in sputum
smears are significantly increased when
sputum is first treated with 5% sodium
hypochlorite (NaOC1), i.e. bleach,
followed by centrifugation.
 NaOC1 treated sputum cannot be used
for culture.

48. Sodium hypochlorite centrifugation technique to
concentrate AFB
Transfer 1–2 ml of sputum (particularly that
which contains any yellow caseous material)
to a sterile container.
Add an equal volume of concentrated sodium
hypochlorite (bleach) solution and mix well.
Leave at room temperature for 10–15
minutes,

49. shaking at intervals to break down the
mucus in the sputum.
Add about 8 ml of distilled water, Mix well.
Centrifuge at 3000 g for 15 minutes.
Using a pipette, remove and discard the
supernatant fluid. Mix the sediment.

50. Transfer a drop of the well-mixed sediment to
a clean scratch-free glass slide.
Spread the sediment to make a thin
preparation and allow to air-dry.
Heat-fix the smear and stain it using the Ziehl-
Neelsen technique.

52.  Preparation of other samples for Z.N. stain:
o Non-purulent fluid specimen: Centrifuge the
fluid and make a smear from a drop of the well-
mixed sediment, (Do not centrifuge a purulent
fluid).
o e.g.
 CSF when tuberculosis meningitis is suspected.
 Effusions samples when tuberculosis is suspected
e.g. (Pleural, Pericardial, peritoneal, and Synovial
Fluids).

53. Steps of Ziehl-Neelsen stain
• Required
1) Carbol fuchsin stain.
2) Acid alcohol 3% v/v.
3) Methylene blue 5 g/l.

55. 1. Cover the fixed smear with carbol fuchsin
stain.
2. Heat the stain until vapour just begins to rise
(i.e. about 60 C). Do not overheat. Allow the
heated stain to remain on the slide for 5
minutes.
3. Wash off the stain with clean water.
4. Cover the smear with 3% v/v acid alcohol for
5 minutes or until the smear is sufficiently
decolorized, i.e. pale pink.

56. 5. Wash well with clean water.
6. Cover the smear with Methylene blue stain
for 1–2 minutes.
7. Wash off the stain with clean water.
8. Wipe the back of the slide clean, and place it
in a draining rack for the smear to air-dry (do
not blot dry).
9. Examine the smear microscopically for AFB,
using the 100X oil immersion objective.
10. AFB will stain bright red, and the
background will stain blue.

62. Interpretation of Ziehl-Neelsen stain
 Results:
• AFB . . . . . . . . . . . . . . . . Red, (straight or
slightly curved rods, occurring singly or in
small groups, may appear beaded).
• Cells . . . . . . . . . . . . . . . . . . . . . . . . .. . Blue.
• Background material . . . . . . . . . . . . Blue.

63.  Reporting of sputum smears:
• 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:
• More than 10 AFB/field . . . . report +++
• 1–10 AFB/field . . . . . . . . . . . report ++
• 10–100 AFB/100 fields . . . . . report +
• 1–9 AFB/100 fields . . . . .report the exact
number.

64.  When no AFB are seen after examining 100
fields:
Report the smear as ‘No AFB seen’. Do not
report ‘Negative’ because organisms may be
present but not seen in those fields examined.
Up to three specimens (one collected as an early
morning specimen) may need to be examined to
detect M. tuberculosis in sputum.

65.  When very few AFB are seen: e.g. when only
one or two AFB are seen, request a further
specimen to examine.
 Tap water and deionized water (using ‘old’
resin) sometimes contain AFB that resemble
tubercle bacilli, and occasionally stained
scratches on a slide can be mistaken for AFB.
Occasionally AFB can be transferred from one
smear to another when the same piece of
blotting paper is used to dry several smears.

66. Quality control of Ziehl-Neelsen stain
At regular intervals, and always when a
new batch of stain is started, two sputum
smears of known high and low AFB
positivity should be stained with the
routine smears to check that the carbol
fuchsin, staining method, and the
microscopical examination of smears are
satisfactory.