DIFFERENTIAL STAIN
Biology and Biotechnology department

Differential stain
group .
separate bacteria into
Differential stain
Gram Stain
Acid-fast Stain
A. GRAM STAIN :



Differentiate bacteria into 2 groups:
Gram positive bacteria
Gram negative bacteria
DIFFERENCES BETWEEN GRAM POSITIVE AND
GRAM NEGATIVE BACTERIA
Gram positive bacteria
Gram negative bacteria
Thick cell wall (many layers of
peptidoglycan)
Thin cell wall ( one layer of
peptidoglycan )
Teichoic acids :
• Lipoteichoic acids
the plasma membrane
• Wallteichoic acids
the peptidoglycan
Absent
link to
link to
 ROLE: Provide rigidity to the cell
wall.
Gram positive bacteria
Gram negative bacteria
No outer membrane
outer membrane (a membranous
structure surround the
peptidoglycan ,part of the capsule) .
 ROLE: Resistance to the
antibiotic
No periplasmic space
periplasmic space (between outer
membrane and the plasma
membrane
Mycolic acid (waxy-lipid)
Found only in acid fast cells
No Mycolic acid
GRAM STAIN COMPONENTS
Like most differential staining procedures has :
Primary stain , mordant and / or selective treatment
(decolorizer), and counter stain .

1. Primary stain : colors the target cells or cell components.
Here the primary stain is crystal violet & the target
cells are the thick-walled bacterial cells.
2. Mordant : react with the primary stain and the target
cells so that the target cells retain the stain (fixation of
stain) .
Here the mordant is Gram's iodine (solution of iodine and
potassium iodide )
3. selective treatment (decolorizing agent ) :
Cause the target cells to retain the primary
stain while removing it from the non-target cells
95% ethanol rinse is used here to remove excess
crystal violet
4. Counter stain is which color every things
wasn't colored by the primary stain
 Safranin is used.
PROCEDURES OF GRAM STAIN











Smear of bacteria.
Stain by crystal violet (30-60 sec)
Washing by D.W
Mordant iodine (60 sec)
Washing
decolorizing agent 95% ethanol drop by drop
Washing
Safranin (30 sec)
Washing
Dry
mirsope
RESULT

Notes :
Ethanol
In (G + ve)
Dehydrates of
peptidoglycan
So CV-I crystals donot
leave
In (G-ve)
Dissolve outer membrane
& holes in peptidoglycan
So CV-I crystals wash out
Examples :
Gram positive bacteria : Bacillus , streptococcus
Gram negative bacteria : E.coli






Never used old culture
Never used sample for patient take antibiotic
(antibiotic distraction of cell wall)
No cell wall ex: Myoplasma
Mycopatrium (g +ve) but have waxy lipid so cannot
stain with gram but stain with acid-fast stain .
Time of decolonization :
over :G+
Glow :GG+
Time of fixation (mordant ) :
over :G+
Glow : no sample remain on the slide after wash
B. ACID-FAST STAIN
Differentiate bacteria into 2 groups:
Acid-fast bacteria:
 retain of the basic stain in the presence of Acid-alchole
 The cell wall of these bacteria have high
concentration of waxy lipid (mycolic acid), macking
simple stain & gram stains useless
(all acid-fast bacteria are gram +)
(Difficult to stain & if stained difficult to remove the
bacteria:
 lose the basic stain when rinsed with Acid-alchole and
usually counterstained to see them
 Don't have waxy lipid

Acid-fast bacteria are pathogenic :
Mycobacterium tuberculosis
tuberculosis
Mycobacterium leprae
leprosy

Acid-fast Stain components:
1. Primary stain is carbol fucsin
2. Decolorizing agent is Acid-alchole
3.Counter stain is methylen blue
PROCEDURES OF ACID-FAST STAIN :










Smear of bacteria
Carbol fucsin + tergitol (wetting agent) cover bacteria
5 mines
if tergitol is not available Carbol fucsin cover bacteria
place slide on pre-warmed hot plate 5 mines
These facility the penetration of waxy lipid
Acid-alchole drop by drop
Washing by D.W
M.B 1-2 mines
D.W
Dry
mirsope
RESULT :