3. Gram staining
• A staining technique used to classify bacteria
• Those that retain the gentian violet are Gram-positive and those that
do not retain it are Gram-negative
9. 4. GRAM SAFRANINE
• Counter stain
• Red colored
• Stains thin walled Gram neg organism
• Pus cells cytoplasm & lobes of nuclei also stain red
10. • Step 1 - Prepare a Smear
• Put some of the material to be stained on a microscope slide,
make thin smear. Allow to air dry.
• Heat fix by gently warming
The Gram Stain Procedure
Watch what happens to the “Bacteria” at each step
“Bacteria”
11. Step 2 - Apply the Primary Stain
Flood the Smear with Crystal Violet
Allow to stand for 1 min
Rinse with water to remove excess stain
12. Step 3 - Apply the Mordant
Flood the Smear with Iodine solution
Allow to stand 1 min
13. Step 4 - Rinse
Rinse with water to remove excess Iodine
14. Step 5 - Decolorize
Drip Decolorizer (Acetone) across the slide about 5
sec
The effluent should appear pale or clear
15. Step 6 - Rinse
Rinse with water to remove excess acetone
16. Step 7 - Counterstain
Flood the slide with Safranin solution
Let stand for 1 minutes
17. Step 8 - Rinse, Dry and Observe
Gram-Positive Gram-Negative
Rinse with water to remove excess stain
Blot dry
Observe under Oil Immersion
18. Choosing a Right Smear
Before choosing a field for
microscopic examination, it is
important to look at the smear
macroscopically
Note that the smear is easily
visible in ordinary light
19. Correct preparation
• Proper smear preparation produce a monolayer of organisms sufficiently
dense for easy visualization but thin enough to reveal characteristic
morphological characteristics.
• Use clean, new glass slides.
• The length of time that crystal violet and Gram’s iodine are left on the
smear is not critical.
• In general, the decolorizing solution is rinsed across the smear until the
decolorizing fluid is no longer blue.
20. Colors makes the Difference in Gram staining
• Bacteria that manage to keep the
original purple are called Gram
positive.
• Bacteria that lose the original
purple dye and can therefore take
up the second red dye are called
Gram negative
21. Report as follows
If no microorganisms are seen in a smear of a clinical
specimen, report “No microorganisms seen.”
If microorganisms are seen, report relative numbers
and describe morphology.
Observe predominant shapes of microorganisms
37. GRAM VARIABLE
Gram variability
Old cultures
Decolorize improperly
Dead and alive bacteria together
Autolytic organisms e.g.
Streptococcus pneumoniae
The smear should be air dried. It should not be heated to speed up drying, because the heat distorts the morphology of bacteria and cells. It should not be placed in front of a fan or waved around the room, because such maneuvers aerosolize material on the slides, including potential pathogens such as Mycobacterium tuberculosis.