GRAM STAINING

MUNAWIR
 LEARNING OBJECTIVES
• To know the procedure of gram staining
• To know the principle difference between
positive and negative gram
 BACKGROUND
• Preliminary step in the initial
characterization and classification of bacteria

• Key procedure in the identification of bacteria

based on staining characteristics

• It is also an important step in the screening

of infectious agents in clinical specimens such
as direct smears from a patient
 OBJECTIVES
• To differentiate between the two major
categories of bacteria: Gram positive and
Gram negative.

• To understand how the Gram stain reaction

affects Gram positive and Gram negative
bacteria based on the biochemical and
structural differences of their cell walls.
HISTORY
• 1882  The Danish
bacteriologist : Hans
Christian Gram
(published in 1884) in
Berlin Hospital
• To discriminate between
pneumococci and
Klebsiella pneumoniae
bacteria in lung tissue
CELL WALL STRUCTURE OF BACTERIA

Gram-positive cell wall Gram-negative cell wall

 COMPONENTS OF GRAM STAINING
• Primary stain (Crystal violet, methyl violet or Gentian
violet)
• Mordant (Gram's Iodine)
• Decolourizer (ethyl alcohol, acetone or 1:1 ethanol-
acetone mixture)
• Counterstain (Dilute carbol fuchsin, safranin or
neutral red)
 EQUIPMENTS
• Bunsen burner
• alcohol-cleaned microscope slide
• Water
• Sengkelit
• Handcloves
• Microscope
 PROCEDURES
Prepare a Slide Smear

• Put on gloves and tie back long hair to

prevent contaminating the bacteria
sample you'll be testing.

• Sterilize a glass microscope slide. If the

glass slide is dirty,
– wash it in soapy water to remove
grease and dirt. D
– disinfect the slide with ethanol, glass
cleaner, or whichever method is
recommended by your laboratory.
PROCEDURES AND PRINCIPLE
– or + Gram ?
+ or - Gram ?
 PROCEDURES
Dispose of materials

• Waste disposal
procedures
– Typically, the liquid in the
staining tray is disposed
of in sealed bottles as
hazardous waste.
– Soak slides in a 10%
bleach solution, then
dispose of them in sharps
containers.
CONCLUSION
 REFERENCES
• Austrian, R. (1960). "The Gram stain and the etiology of lobar pneumonia, an historical note". Bacteriol. Rev. 24 (3):
261–265. PMC 441053. PMID 13685217.

• Gram, HC (1884). "Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten". Fortschritte der
Medizin (in German) 2: 185–189..
English translation in: Brock, T.D. (1999). Milestones in Microbiology 1546–1940 (2 ed.). ASM Press. pp. 215–218.
ISBN 1-55581-142-6..
Translation is also at: Brock, T.D. "Pioneers in Medical Laboratory Science: Christian Gram 1884". Hoslink. Retrieved
2010-07-27.

• Rao Shiradar. Gram’s staining. Dept. of microbiology JVVVC. Davenger . 2004.

http://www.microrao.com/micronotes/pg/Gram%20stain.pdf

• Virtual Amrita Laboratory. Gram Stain Technique. New Delhi 2014.

http://amrita.vlab.co.in/?sub=3&brch=73&sim=208&cnt=1

• Mims, c et all. The bacteria in Medical microbiology. Toronto. Elsevier. 2004. P. 11-12

• Kleuring, G. Gram Stain Technique in protocol Molecular Cell Physiology 2001.

http://www.falw.vu/~microb/Protocols/General_protocols/Gram-Kleuring.pdf

• Hardy, J. Gram’s Serendipitous Stain. Santa Maria. 2002 http://hardydiagnostics.com/articles/Hans-Christian-

Gram.pdf

• UPHS. How to do Gram Stains. 2004. http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/Gram2.htm

THANK YOU