GRAM STAINING

AIM:

To perform Gram stain in the given smear.

PRINCIPLE:

Gram positive cells which have a thicker cell wall retain the dye-I 2 complex , In Gram negative
cells the dye-I2 complex will comeout due to thinner cell wall and take up the counterstain.

THEORY BEHIND GRAM STAINING:

• Cell wall theory: Cell wall of Gram positive bacteria (peptidoglycan layer) are 40 times thicker
than those of Gram negative cells, hence they retain the dye-iodine complex.

• Lipid Content Theory: The high lipid content of Gram negative bacteria is washed out by the
organic solvents like acetone after decolourisation and permits the dye iodine complex to come
out and makes them permeable to the secondary dye

• Cytoplasmic pH Theory: Gram positive cells have more acidic protoplasm than the Gram
negative and hence retain the basic primary dye more strongly than the Gram negative one.

PROCEDURE:

1. A smear is made, air-dried/ heat-fixed in a grease free slide.

2.Primary staining: with pararosaniline dye such as crystal violet / methylviolet / gentian
violet. Leave it for 1minute.

3.Mordant - Gram's iodine which fixes the primary stain is used and Leave it for one minute.
4. Decolourisation- with organic solvent such as alcohol (1min.) / acetone (2sec.)
5. Counterstaining - with Dilute carbol fuchsin / Safranin / neutral red. Leave it for one min.

6. The slide is air dried and observed it under oil immersion.

7. The Gram stain differentiates bacteria into two groups. Gram positive bacteria are those that
resist decolourisation and retain the primary stain appearing violet. Gram negative bacteria
are decolourised by the organic solvents and take up the counterstain, appearing pink.
 GRAM STAINING - GPC

AIM:

To perform Gram stain in the given smear.

OBSERVATION:

1 Colour Purple

2 Shape Spherical

3 Arrangement clusters

INFERENCE:

The given smear contains Gram positive cocci arranged in clusters.

EXAMPLES: Staphylococcus aureus, Staph epidermidis.

 GRAM STAINING - GNB

AIM:

To perform Gram stain in the given smear.

OBSERVATION:

1 Colour Pink

2 Shape Rod

3 Arrangement Scattered

INFERENCE:

The given smear contains Gram negative bacill with Scattered arrangement.

EXAMPLES: Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Klebsiella

pneumoniae, Proteus mirabilis

GRAM STAINING - MIXTURE

AIM:
 To perform Gram stain in the given smear.

OBSERVATION:

1 Colour Purple Pink

2 Shape Spherical Rod

3 Arrangement Clusters Scattered

INFERENCE:

The given smear contains both Gram positive cocci arranged in clusters and Gram negative
bacilli in Scattered pattern.

FREQUENTLY ASKED QUESTIONS IN GRAM STAINING

1. Who invented Gram stain?

Hans Christian Gram in 1884.
2. Explain the original formulation of Gram staining.

The original formulation was Gentian violet, Lugol’s iodine, absolute alcohol and Bismark
brown.

3. Give example for differential stain.

Gram stain, acid fast stain, metachromatic granule staining.

4. State why Gram stain is said to be a differential stain.

Gram’s stain is said to be a differential stain because it differentiates bacteria as Gram positive
bacteria (appears violet) and Gram negative bacteria (appears pink).

5. Why bacteria are classified as Gram positive or Gram Negative?

Based on the cell wall constituents and differences in the thickening of the peptidoglycan layer, those
bacteria appearing violet are called Gram positive (retaining the primary dye) and those bacteria
appearing pink are called Gram negative (take up the counter stain).

6. What are the various steps /procedure in Gram staining?

 Primary staining
 Addition of Mordant
 Decolorization,
 Counterstaining

7. What are the various primary stains that can be used in Gram staining?

Crystal violet, Methyl violet and Gentian violet

8. What is the function of Gram’s iodine?

It acts as a mordant.

9. What do you mean by mordant?

Fixing agent

10. What is the role of Grams iodine in Gram staining?

Iodine makes the cytoplasm more acidic and acts as a mordant (fixing agent. It thus helps
to fix the stain in the bacterial cell and forms the dye-iodine complex.

11. What are the various decolourisers that are used in Gram staining?
 95% Ethyl Alcohol, Acetone, 1% Acid alcohol, Aniline, Acetone -alcohol mixture (1:1),
iodine acetone.

12. What are the various counter stains that are used in Gram staining?

Dilute carbolfuchsin, Safranine, Neutral red and if person is color blind, Bismark brown
may be used.

13. What is the important step in Gram staining?

Decolourisation.

14. What happens if you over decolourise?

Over-decolourization can result in Gram positive bacteria appearing Gram negative.

15. What happens if you under decolourise?

Under-decolourization can result in Gram negative bacteria appearing Gram positive.

16. Which part of the bacteria actually gets stained?

It is the cytoplasm (especially the nucleic acid) that gets stained and not the cell wall. Presence of
an intact cell wall is important for retaining Gram positivity. Cell wall deficient forms such as
Mycoplasma and L forms are Gram negative.

17. Explain the mechanism of Gram staining.

Gram positive – acidic protoplasm - retain basic primary dye. Peptidoglycan of Gram-positive
bacteria thick – retain the dye iodine complex. High lipid content of Gram-negative bacteria
washed out during decolourisation and takes up the counter stain appearing pink.

18. Which are the bacteria or bacterial component that can’t be stained by Gram stain?

• Extremely slender bacteria such as Treponema.

• Cells containing waxy substances impermeable to stain such as Mycobacterium.
• Minute intracellular bacteria such as Chlamydia and Rickettsia.
• Cell organelles such as capsule, spore, flagella etc.,
19. What are the differences between cell wall of Gram positive and Gram negative
bacteria?

 Gram positives have a thick peptidoglycan layer in the cell wall when compared
to Gram negatives which possess a thinner peptidoglycan layer.
 Teichoic acid is present in Gram positive cellwall while it is absent in Gram
negative cell wall.
 Lipopolysaccharide is absent in Gram positive cell wall and is present in Gram
negative cell wall.

20. Give example for Gram positive cocci

Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae.

21. Name the Gram positive cocci arranged in clusters.

Staphylococcus aureus

22. Name the Gram positive cocci arranged in chains.

Streptococcus pyogenes

23. Name the Gram positive lanceolate shaped cocci arranged in pairs.

Streptococcus pneumonia

24. Give example for Gram positive cocci arranged in pairs and short chains.

Enterococcus specie

25. Name few Gram positive bacilli.

Corynebacterium diphtheriae, Bacillus anthracis, Clostridium spp.

26. Name few Gram negative cocci.

Neisseria meningitidis, Neisseria gonorrhoeae.

27. Name few Gram negative bacilli.

Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella spp.,

Shigella spp., etc.,

28. Name a comma shaped Gram negative bacilli.

Vibrio cholera
29. Name few intracellular Gram negative cocci arranged in pairs.

Neisseria meningitidis

Neisseria gonorrhoeae

30. Name few anaerobic Gram positive cocci

Peptococci, Peptostreptococci.

31. Name anaerobic Gram negative cocci.

Veillonella.

32. Name an anaerobic Gram negative bacilli.

Bacteroides

33. How do spores appear in Gram staining?

The spores do not take any colour and appear unstained in Gram staining.

34. Name some Gram positive yeast cells.

Cryptococcus neoformans, Candida albicans.

35. Name the bacteria that are used as the positive and negative controls for Gram stain?

Positive control: Staphylococcus aureus

Negative control: Escherichia coli

36. What are the conditions when Gram positive bacteria can appear Gram negative?

• When over-decolorized by either prolonged exposure to decolourizer.

- When cell wall gets damaged by exposure to lysozyme or cell wall acting antibiotics.
such as Penicillin and Cephalosporins
• Old cultures, where cell wall is weakened or action of autolytic enzymes

37. What are Gram variable bacteria?

Gram variable bacteria are those Gram positive bacteria that have lost their cell wall
integrity because of antibiotic treatment, old age or action of autolytic enzymes. These
 changes allow methyl violet to come out of the cell wall during process of decolourising
resulting in some cells staining pink and others staining purple.

38. What are the applications of Gram staining?

a) Rapid presumptive diagnosis of diseases such as bacterial meningitis.

b) Selection of empirical antibiotics.
c) Selection of suitable culture media.
d) Screening of quality of clinical specimens, such as sputum that should contain many pus cells
and few epithelial cells.
e) For quantitative estimation of bacteria in urine specimens:

 Identifies urine specimens that contain bacteria greater than 105 cfu/ml of
urine.
 Presence of at least 1 organism/oil immersion field correlates with
significant bacteriuria (105 cfu/ml).

f) Appreciation of morphology and types of bacteria in a clinical specimen.