Microbiology

1. SMEAR PREPARATION &
GRAM STAINING
Dr.priyadharshini.S ,
1st year postgraduate ,
Department of microbiology ,
Chettinad hospital and research institute ,

2. SMEAR PREPARATION
SMEAR should be spread evenly covering an area of about 15 - 20mm diameter on a slide.
The technique used to make smears from different specimens are as follows:
■ Purulent specimen: using a sterile wire loop, make a thin preparation.donot centrifuge a purulent fluid eg 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.
■ Culture: Emulsify a colony in sterile distilled water and make a thin preparation on a slide, when a broth
culture transfer a loop ful to a slide and make a thin preparation.
■ Sputum : use a piece of clean stick to transfer and spread purulent and caseous material on a slide. Soak
the stick in a phenol Or hypochlorite disinfectant before discarding it.
■ Swabs: Roll the swab Ona slide. This is particularly important when looking for intracellular bacteria such as
N.gonorrhoea ( urtheral, cervical, eye swab). Rolling the swab avoids damaging the pus cells.
■ Faeces: Use apiece of clean stick to transfer pus and mucus to a slide decontaminate the stick before
discarding it.spread to make a thin preparation.

3. DRYING AND FIXING SMEARS
■ After making a smear,leave the slide in a safe place For the
smear to air dry,
■ When a smear requires urgent staining, it can be dried
quickly using gentle heat
■ The purpose of fixation is to preserve microorganisms and to
prevent smear being washed from slides during staining.
■ Smears are fixed by heat, alcohol, or occasionally by other
chemicals.

4. HEAT FIXATION:
■ This is widely used but can damage organisms and alter their
staining reactions especially when excessive heat is used. Heat
fixation also damages leucocytes and is therefore unsuitable for
fixing smears which may contain intracellular organisms such as
N. Gonorrhoeae and N. Meningitidis.
■ When used, heat fixation must be carried out with care. The
following technique is recommended:
■ Allow the smear to air-dry completely.
■ Rapidly pass the slide, smear uppermost, three times through the
flame of a spirit lamp or pilot flame of a Bunsen burner.
■ Note: After passing the slide through the flame three times, it
should be possible to lay the slide on the back of the hand without
the hand feeling uncomfortably hot. When this cannot be done,
too much heat has been used.

5. 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 diplococci.
■ Alcohol fixation is more bactericidal than heat ( eg: M.tuberculosis is rapidly killed in
sputum smears after applying 70% alcohol)
■ A method of alcohol fixation is as follows:
1. Allow the smear to air dry completely.
2. Depending on the Type of smear, alcohol fix as follows:
– For the detection of intracellular gram negative diplococci (N.gonorrhoeae or

6. CHEMICAL FIXATIVES
■ Other chemicals are some times necessary to fix smears which contains
particularly dangerous organisms to ensure all the organisms are killed
eg 40g/l potassium permaganate is recommended for fixing smears
which may contain Anthrax bacilli.
■ Formaldehyde vapour is sometimes recommended for fixing smears
which may contain MYCOBACTERIUM species.
■ Formaldehyde fixed smears however tend to stain poorly and the
chemical itself is toxic with an injurious vapour.

7. GRAM STAINING
•Hans Christian
Joachim Gram
( 13 September 1853 –
14 November
1938)

8. CORNER STONE OF BACTERIAL INDENTIFICATION
■ Danish physician Hans Christian Gram developed the Gram
staining method in 1884.
■ Gram staining is still the cornerstone of bacterial
identification and taxonomic division. Besides demonstrating
the morphology of bacteria ,the gram stain can be used to
divide most bacterial species into 2 groups.
■ Those that take up the basic dye ( crystal violet) is GRAM
POSITIVE
■ Those that allow the crystal violet dye to washout easily with
decolourizer (Alcohol or Acetone) is GRAM NEGATIVE
■ Gram-positive bacteria- stains Darkpurple
■ Gram-negative bacteria-stains red/pink

9. PRINCIPLES OF GRAM STRAINING
■ CELL WALL THEORY:The differences in Gram-positive and Gram-negative bacteria
cell wall composition account for the Gram staining differences. Gram-positive cell wall
contains a thick layer of peptidoglycan with numerous teichoic acid cross-linking,
which resists decolorization.
■ PH THEORY: In aqueous solutions, crystal violet dissociates into CV+ and Cl – ions
that penetrate through Gram-positive and Gram-negative cell walls. The CV+ interacts
with negatively charged componeIn contrast, the nts of bacterial cells, staining the cells purple.
When added, iodine (I- or I3-) interacts with CV+ to form large crystal violet-iodine
(CV-I) complexes within the cytoplasm and outer layers of the cell.
■ The decolorizing agent (ethanol or an ethanol and acetone solution) interacts with the
lipids of both gram-positive and gram-negative bacteria membranes.
■ The outer membrane of the Gram-negative cell is lost from the cell, leaving the thin
peptidoglycan layer exposed. With ethanol treatment, gram-negative cell walls become
leaky and allow the large CV-I complexes to be washed from the cell.
■ The highly cross-linked and multi-layered peptidoglycan of the gram-positive cell
is dehydrated by the addition of ethanol. Thus ethanol treatment traps the large CV-I
complexes within the cell.
■ After decolorization, the gram-positive cell remains purple. In contrast, the gram-

10. Cell wall of Gram-positive and Gram-negative
Bacteria

12. All cocci are gram positive
EXCEPT:
1. Meningococci
2. Gonococci
3. Veilonella
4. Moraxella
All bacilli are gram negative
Except:
1. MYCOBACTERIUM
2. ANTHRACIS BACILLUS
3. CLOSTRIDIUM SP
4. DIPTHERIAE CORYNEBACTERIUM
5. NOCARDIA
6. ACTINOMYCETES
7. LISTERIA
8. DIPTHEROIDS

13. STEPS OF GRAM STAINING
■ Fixation of clinical materials made on a slide from bacterial culture
or specimen is air dried and then heat fixed.
■ STEP 1:Application of the primary stain (crystal violet) for 1
minute. Then he slide is rinsed with water.Crystal violet is a dark
blue to purple dye. It stains all cells blue/purple.
■ STEP 2:Application of mordant: The Gram’s iodine solution
(mordant) is Poured over the slide for 1 minute.then the slide is
rinsed with water.Grams iodine form a crystal violet-iodine (CV-I)
complex; all cells continue to appear blue.
■ STEP 3:Decolorization : Pouring few drops of decolourizer to the
smear eg: acetone for 1-2sec or ethyl alcohol 20-30sec or Acetone
alcohol for 10sec.slide is immediately rinsed with water.The
decolorization step distinguishes gram-positive from gram-negative
cells. The organic solvent such as acetone or ethanol extracts the blue
dye complex from the lipid-rich, thin-walled gram-negative bacteria
to a greater degree than from the lipid-poor, thick-walled, gram-

14. Note: Decolorization is the most crucial step of gram staining.if the
decolourizer is Poured for more times even gram positive bacteria lose
color (Over Decolorization)
And if poured less timethe gram negative bacteria donot lose the
primary color of primary stain properly ( under Decolorization)
■ STEP 4:
Application of counterstain (safranin) is added for 1
minute.then the slide is rinse with water. The red dye
safranin stains the decolorized gram-negative cells
red/pink; the gram-positive bacteria remain blue.
The slide is dried and then examined under oil
immersion objective.

15. • After performing a gram stain, the technician should first determine whether
the Gram stain is adequate. In an appropriately stained biological specimen,
the nuclei of neutrophils are red. If the nuclei are blue, the decolorization is
insufficient.
Results
■ Gram-negative bacteria will stain pink/red and
■ Gram-positive bacteria will stain blue/purple.

16. REAGENTS:
violet dye:
■ Crystal violet or methyl violet is used as a concentration of 0.5 to
2 %.
1. Crystal violet 10g
2. Absolute alcohol 100ml
3. Distilled water 1litre
■ Dissolve the dye in the alcohol, filter through filter paper and
add to the water.

17. ■ However, gram positive staining can be strengthened by the addition of sodium
bicarbonate or ammonium oxalate as in the following solutions:
1. Kopeloff & Beerman’s stain :
solution A: methylviolet 10g
Distilled water 1litre
Solution B: sodium bicarbonate 50g
. Distilled water 1 litre
Shortly before use mix 30 volumes of solution A with 8 volume of solution B
It is a disadvantage of this mixture that it tends to precipitate within a few days and
so cannot be kept.
2. Preston & Morrells stain:
. Crystal violet 20g, methylated spirit 200ml, Ammonium oxalate
1% in water 800 ml

18. IODINE SOLUTION
■ To obtain iodine in aqueous solution, potassium iodide or sodium hydroxide
must be added.
■ The more alkaline solution with sodium hydroxide is thought to give slightly
stronger gram positive staining.
■ Gram’s iodine (lugol’s):
. Iodine 10g
Potassium iodide 20g
Distilled water 1 litre
Kopeloff & Beerman’s iodine:
Iodine 20 g
Sodium hydroxide (4%NAOH) 100ml
Distilled water 900ml

19. DECOLOURIZER:
■ ACETONE:
1. this is the fastest and most specific decolourizer.
2. It is applied to the smear for only 2-3 Seconds and to sections for a
second or 2 longer.
3. It’s rapidity is an advantage when only a single slide is to be stained.
4. However the short period of exposure is difficult to control when many
slides have to be stained simultaneously.

20. ABSOLUTE ALCOHOL(100%ETHANOL)
■ This acts more slowly than acetone and should be applied and reapplied
for about 1 min until on tilting the slide from slide to slide .
ACETONE ALCOHOL:
■ This is a mixture of 1 volume of acetone with 1 volume of 95% ethanol.
■ It requires for about 10 Seconds.
IODINE ACETONE:
■ Preston & morrell have shown that the addition of a small concentration
of iodine to acetone slows it’s rate of Decolorization without reducing its
specificity.
■ With 0.35%iodine the exposure can be lengethed from about 2 seconds to
30 seconds or longer.

21. COUNTERSTAIN
1. Dilute carbol fuchsin:
■ This stain when applied gives the strongest red staining
■ However the colouration may be so dark that some gram negative bacteria mY be
difficult to distinguish from gram positive ones.
■ It’s is better to use one of the following weaker counterstain.
1. BASIC FUCHSIN COUNTERSTAIN : It is applied for 10-30 sec this is
recommended for general use.
2. NEUTRAL RED COUNTERSTAIN: applied for 2-4 min this is recommended
for demonstrating gonococci and other intracellular gram negative bacteria.
3. SAFRANIN COUNTERSTAIN: Safranin 0.5 % in distilled water

22. 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. Also, whether
the organisms are intracellular.
■ Presence and number of pus cells
■ Presence of yeast cells and epithelial cells.

23. USES OF GRAM STAIN:
■ To differentiate bacteria into gram positive & gram negative
■ For identification: gram staining from bacterial culture gives an idea to put the
corresponding biochemical tests for further identification of bacteria.
■ To start empirical treatment: gram stain from the 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 antibiotics
can be started early before 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.
■ Yeasts: in addition to stain the bacteria, gram stain is useful for staining certain
fungi such as Candida and Cryptococcus ( appear gram positive)
■ Quality of specimen: gram stain helps on screening the quality of the sputum
specimen before processing it for culture. Presence of More pus cells and less
epithelial cells indicates good quality specimen.

24. MODIFICATIONS OF GRAM STAINING:
■ KOPELOFF & BEERMAN’S MODIFICATION:
Primary stain and counter stain used are methylviolet and basic fuchsin
respectively.
■ JENSEN’S MODIFICATION:
This method involves use of absolute alcohol as decolourizer and neutral
red as counterstain
It is useful for meningococci and gonococci.
■ BROWN &BRENN MODIFICATION:
This is used for Actinomycetes.

25. LIMITATIONS
■ The sensitivity of the Gram stain procedure is low. Sometimes, you
may fail to see the organism in Gram Stain smear, but the same
clinical specimen may yield organisms when cultured. To be visible
on a slide, organisms that stain by the Gram method must be present
in about 104 to 105 organisms per milliliter of centrifuged fluid.
■ Gram staining technique is not recommended for spirochetes and
mycobacteria. Mycobacteria stain weakly with gram stain, and
bacteria such as Mycoplasma, Rickettsiae, Chlamydiae do not take
up the dyes used in Gram stain or are too small to be seen with light
microscopy.
■ Not all bacteria can be seen in the Gram stain. This is the list of
medically important bacteria that can be not seen in Gram-stain.

26. Nearly all clinically important bacteria can be visualized using the
Gram staining technique, the only exceptions being those
organisms;
• That exists almost exclusively within host cells (intracellular
bacteria) (e.g., Chlamydia)
• Those that lack a cell wall (e.g., Mycoplasma)
• Those of insufficient dimensions to be resolved by light
microscopy (e.g., spirochetes)

27. Name Reason
Alternative
Microscopic
Approach
Chlamydiae, including
C. trachomatis
Intracellular; very small
Inclusion bodies in the
cytoplasm
Legionella pneumophila
Poor uptake of red
counterstain
Prolong time of
counterstain
Mycoplasma
pneumophila
Poor uptake of red
counterstain
None
Mycobacterium, includin
g
M. tuberculosis
Too much lipid in cell
wall so dye cannot
penetrate
Acid-fast stain
Rickettsiae Intracellular; very small
Giemsa or other tissue
stains
Treponema pallidum Too thin to see
Dark-field microscopy or

28. QUALITY
COUNTROL
■ Always check new batches of stain and reagents for correct staining
reactions using a smear containing known Gram-positive and Gram-
negative organisms.
Variations in Gram Reaction
■ Various factors influence the results of Gram staining. Sometimes the
result might be entirely different than you have anticipated.
■ Gram-positive bacteria may lose their ability to retain crystal violet
and stain Gram negatively for the following reasons:
– cell wall damage of bacteria due to antibiotic therapy or excessive
heat fixation of the smear.
– over- decolorization of the smear
– use of an old Iodine solution that is yellow in color instead of
brown (always store in a brown glass or other light opaque
containers).
– preparation of smears from old culture.
A thick smear will require more decoloration than a thin smear

29. Organism
Classic
Presentation
Variant
Presentation
Comments
Streptococccus
pneumoniae
Gram-positive, lancet-
shaped, diplococci
Elongated cocci,
resembling short bacilli
May be misinterpreted
as mixed organisms;
over-decolorized cells
may be mistaken for
gram-negative
coccobacilli.
Acinetobacter spp.
Gram-negative
coccobacilli
Gram-negative cocci;
gram-variable staining
is common
May be mistaken
for Neisseria spp. and
reported as gram-
negative cocci; search
the smear to find some
organisms that
demonstrate elongated
forms, which are not
seen in Neisseria.
Yeast,
especially Cryptococc
us neoformans
Gram-positive round or
oval cells with budding
Gram-variable cells
May be mistaken for
artifacts; size and
shape distinguish them
from bacteria

30. Clostridium
perfringens
Boxcar-
shaped gram-
positive bacilli
Gram-variable or
Gram-negative
bacilli
Maybe mistaken
for gram-negative
bacilli; the boxcar
shape is a clue
that the organism
is gram-positive;
other Clostridia an
d Bacillus spp. May
also appear
similar.

31. THANKYOU