Presentation on Gram staining for Microbiology students

1. -PREETI SHARMA

2. INTRODUCTION:
• Gram staining is a differential
stain.
• Helps to differentiate between
gram positive and gram
negative structures

3. HANS CHRISTIAN GRAM
Aniline Gentian violet
Lugol’s iodine
Absolute alcohol
Bismarck brown

6. CELL WALL THEORY:
Gram positive cell wall has a thick
peptidoglycan layer with tight cross
linkages.
Whereas the gram negative cell wall
has lipopolysacharide which gets
disrupted easily by the decolorizer.

7. PH THEORY
Cytoplasm of gram positive
bacteria is more acidic hence
retain the basic dye for longer
time.

8. MAGNESIUM
RIBONUCLEATE THEORY
Present in cell membrane of gram
positive bacteria and it helps to
retain the primary dye.

9. PROCEDURE
FIXATION: The smear
made on the slide from the
bacterial culture/ specimen is
air dried and then heat fixed.

13. GRAM POSITIVE BACTERIA
COCCI
• STAPHYLOCOCCUS
• STREPTOCOCCUS
• ENTEROCOCCUS
• MICROCOCCUS

15. BACILLI
• CORYNEBACTERIUM
• CLOSTRIDIUM
• LISTERIA
• BACILLUS

17. GRAM NEGATIVE BACTERIA
COCCI
• NEISSERIA
• MORAXELLA
• BRANHAMELLA

19. BACILLI
• E.COLI
• KLEBSIELLA
• PSEUDOMONAS
• PROTEUS
• SALMONELLA
• SHIGELLA

21. GRAM VARIABLE BACTERIA
• GARDNERELLA
• LISTERIA
• ACTINOMYCES
• ARTHROBACTER
• PROPIONIBACTERIUM

23. MODIFICATIONS

24. KOPELOFF & BEERMAN’S
PRIMARY STAIN: Methyl violet with Sodium
bicarbonate.
MORDANT: Iodine with Sodium hydroxide
DECOLORIZER: Acetone
COUNTERSTAIN: Basic fuschin

25. PRESTON MORELL
PRIMARY STAIN: Ammonium oxalate crystal violet
MORDANT: Lugol’s 1% Iodine
DECOLORIZER: Iodine-acetone
COUNTERSTAIN: Dilute carbol fuschin

26. JENSEN
PRIMARY STAIN: 0.5% Methyl Violet
MORDANT: Lugol’s Iodine
DECOLORIZER: Absolute alcohol
COUNTERSTAIN: Neutral red

27. WEIGERT’S
PRIMARY STAIN: Carbol Gentian Violet
MORDANT: Gram’s Iodine
DECOLORIZER: Aniline Xylol
COUNTERSTAIN: Dilute carbol fuschin

28. USES OF GRAM STAIN

29. IDENTIFICATION
Gram Positive
& Gram Negative
Fastidious
Organism
Anaerobes
Candida,
Cryptococcu
s

30. Quality
of
specimen
especially
sputum
sample.

31. To start empirical treatment

32. REFERENCES;
• Koneman's Color Atlas and Textbook of Diagnostic
Microbiology
• Mackie & McCartney Practical Medical Microbiology, JG
Collee
• Ananthanarayan and Paniker's Textbook of Microbiology
• Essentials of Medical Microbiology by Apurba Sankar Sastry
and Sandhya Bhat
• American Society for Microbiology Conference for
Undergraduate Educators (ASMCUE). 2005. [cited August
10, 2005]

33. THANK YOU