1. Biology and Biotechnology department
Differential stain
Biology and Biotechnology department

2. Differential stain Gram Stain Acid-fast Stain
Differential stain separate bacteria into group .
Differential stain
Gram Stain
Acid-fast Stain

3. Gram Stain : Differentiate bacteria into 2 groups:
Gram positive bacteria
Gram negative bacteria

4. differences between gram positive and gram negative bacteria
Gram positive bacteria
Thin cell wall ( one layer of peptidoglycan )
Thick cell wall (many layers of peptidoglycan)
Absent
Teichoic acids :
Lipoteichoic acids link to the plasma membrane
Wallteichoic acids link to the peptidoglycan
ROLE: Provide rigidity to the cell wall.

5. Gram negative bacteria
Gram positive bacteria
outer membrane (a membranous structure surround the peptidoglycan ,part of the capsule) .
ROLE: Resistance to the antibiotic
No outer membrane
periplasmic space (between outer membrane and the plasma membrane
No periplasmic space
No Mycolic acid
Mycolic acid (waxy-lipid)
Found only in acid fast cells

7. 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 )

8. 95% ethanol rinse is used here to remove excess crystal violet
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.

9. 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
Safranin (30 sec)
Dry
mirsope

10. result

13. Gram positive bacteria : Bacillus , streptococcus
Notes :
Examples :
Gram positive bacteria : Bacillus , streptococcus
Gram negative bacteria : E.coli
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

14. 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 G-
low :G G+
Time of fixation (mordant ) :
low : no sample remain on the slide after wash

15. 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

16. 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

17. :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

18. Result :