Muhammad ali mushtaq Gram Stain lecture.ppt

Gram Staining is a widely used microbiological technique for the classification and differentiation of bacteria into two main groups: Gram-positive and Gram-negative. Developed by the Danish bacteriologist Hans Christian Gram in 1884, the method is based on the structural differences in the bacterial cell walls.

The Gram stain involves four key steps:

Crystal violet application: First, a bacterial smear is stained with crystal violet, a basic dye that penetrates the cell walls of both Gram-positive and Gram-negative bacteria, turning them purple.

Iodine treatment: After the initial staining, iodine, which acts as a mordant, is applied. It forms a crystal violet-iodine complex inside the bacterial cells, enhancing the dye’s retention.

Decolorization: The critical differentiating step involves washing the cells with alcohol or acetone. This step decolorizes Gram-negative bacteria while Gram-positive bacteria retain the purple stain. Gram-positive bacteria have a thick peptidoglycan layer in their cell walls, which traps the crystal violet-iodine complex. In contrast, Gram-negative bacteria have a thinner peptidoglycan layer and an outer lipid membrane, which the alcohol dissolves, allowing the complex to wash out.

Counterstaining: To visualize the Gram-negative bacteria, a counterstain, typically safranin or fuchsine, is applied. This turns Gram-negative bacteria pink or red, while Gram-positive bacteria remain purple.

1. Gram Stain

2. •In 1884 the Danish microbiologist Hans
Christian Gram discovered that crystal violet
irreversibly stains certain bacteria but can be
washed from others. The dye has been widely
used ever since for the Gram stain technique,
which identifies bacteria as gram-positive (the
stain is retained) or gram-negative (the stain is
washed).

3. Staining of BacteriaStaining of Bacteria
Bacteria cells Bacteria cells are are almost almost colorlesscolorless and and
transparenttransparent
A staining technique is often applied to the A staining technique is often applied to the
cells to cells to color themcolor them → →
Their Their shapeshape and and size size can be easily can be easily
determined under the microscope. determined under the microscope.

4. Gram staining a Important Technique
•A staining technique used to classify bacteria;
bacteria are stained with crystal violet and
then treated with Gram's solution; after being
decolorized with alcohol and treated with
safranin and washed in water, those that
retain the crystal violet are Gram-positive and
those that do not retain it are Gram-negative

5. Gram-positive bacteria
• Have a thick peptidoglycan layer surrounds the cell.
•The stain gets trapped into this layer and the bacteria turned
purple.
•Retain the color of the primary stain (crystal violet) after
decolorization with alcohol
Gram-negative bacteria
•have a thin peptidoglycan layer that does not retain crystal
violet stain.
•Instead, it has a thick lipid layer which dissolved easily upon
decoulorization with Aceton-Alcohol.
•Therefore, cells will be counterstained with safranin and
turned red.

6. Gram’s +ve Bacteria Gram’s -ve Bacteria

7. The Cell walls differ…

8. Organizing the Staining Bottles

9. 9
Crystal violet
↓
Iodine
↓
Alcohol
↓
Safranin

10. Method
•Fix the dried smear.
•Cover the fixed smear with crystal violet stain for
30–60 seconds.
•Rapidly wash off the stain with clean water.
•Tip off all the water, and cover the smear with
Lugol’s iodine for 30–60 seconds.
•Wash off the iodine with clean water.
•Decolorize rapidly (few seconds) with acetone–
alcohol. Wash immediately with clean water.

11. •Cover the smear with neutral red or safranin
stain for 2 minutes.
•Wash off the stain with clean water.
•Wipe the back of the slide clean, and place it in a
draining rack for the smear to air-dry.
•Examine the smear microscopically, first with the
40 objective to check the staining and to see the
distribution of material, and then with the oil
immersion objective to report the bacteria and
cells.

12. 12
Gram +veGram +ve
S.aureusS.aureus
Gram –veGram –ve
E.coliE.coli
Step 1:Step 1: Crystal Violet
Step 2:Step 2: Gram’s IodineGram’s Iodine
Step 3: Decolorization Step 3: Decolorization
(Aceton-Alcohol)(Aceton-Alcohol)
Step 4:Step 4: Safranin RedSafranin Red

13. Step 1

14. Step 2

15. Step 3

16. Step 4

17. Step 5

18. Step 6

19. Step 7

20. Result
•Gram positive bacteria . . . . . . . . . . . . Dark purple
•Yeast cells . . . . . . . . . . . . . . . . . . . . . . Dark purple
•Gram negative bacteria . . . . . . . . .Pale to dark
red
•Nuclei of pus cells . . . . . . . . . . . . . . . . . . . . . Red
•Epithelial cells . . . . . . . . . . . . . . . . . . . . . Pale red

21. Reporting Gram smears
The report should include the following information:
● Numbers of bacteria present, whether many,
moderate, few, or scanty.
● Gram reaction of the bacteria, whether Gram positive
or Gram negative.
● Morphology of the bacteria, whether cocci, diplococci,
streptococci, rods or coccobacilli. Also whether the
organisms are intracellular.
● Presence and number of pus cells.
● Presence of yeast cells and epithelial cells.

22. Colors makes the Difference in Gram
staining
•Bacteria that manage to
keep the original purple
dye have only got a cell
wall - they are called Gram
positive.
•Bacteria that lose the
original purple dye and can
therefore take up the
second red dye have got
both a cell wall and a cell
membrane - they are
called Gram negative.

23. Observe with Caution to differentiate

25. Mixed Flora of Staphylococcus and
Streptococcus differentiates morphology

26. Stains Several Fungi

27. Gram-negative Pseudomonas aeruginosa
bacteria (pink-rods).

28. (Bacillus anthracis),

29. Streptococcus pneumonia

30. Gram stain of Neisseria gonorrhoea

31. Streptococcus pneumoniae in Sputum

32. Clostridium difficile as seen by Gram
staining

33. Corynebacterium diptheria as seen in Gram
staining

34. Neisseria meningitides as seen in Gram
staining

35. Nocardia spp seen in Gram Staining

36. Gram Stained Actinomyctes spp

37. Gram Stained Cryptococcus neoformans

38. Variations in Gram reactions
•Gram positive organisms may lose their ability to
retain crystal violet and stain Gram negatively for
the following reasons:
– Cell wall damage due to antibiotic therapy or
excessive heat-fixation of the smear.
– Over-decolorization of the smear.
– Use of an iodine solution which is too old, i.e.
yellow instead of brown in colour (always store in
a brown glass or other light opaque container).
– Smear has been prepared from an old culture.

39. •Gram negative organisms may not be fully
decolorized and appear as Gram positive
when a smear is too thick.

40. Control
•Always check new batches of stain and
reagents for correct staining reactions using a
smear containing known Gram positive and
Gram negative organisms.