Gram stain by manoj

1. GRAM’S STAIN
Mr. Manoj Mahato

2. HISTORICAL
BACKGROUND
• Hans Christian Joachim Gram, Danish bacteriologist and
physician.
 Developed Gram staining technique in 1883 and published his
findings in 1884 in Friedlander’s Journal.
 Was working with respiratory disease in lung tissue from
cadaver at Municipal Hospital Berlin and his accidental
spillage of lugol’s iodine over lung tissue sections led to the
development of Gram Staining.
 While examining the lung tissue from the patients who had
died of pneumonia, he observed that certain stains were
preferentially taken up and retained by bacterial cell.

3. •His initial work with this staining process was performed on
Kleibsella pneumoniae and Streptococcus pneumoniae.
•He did not use a counter stain in his procedure. It was a few
years later, that the German pathologist Carl Weigert (1845-
1904) from Frankfurt, added a final step of staining with
safranin.

4. STAINING
It is the artificial coloration of a substance
to facilitate examination of tissue,
microorganism or other cells under
microscope .

5. Classification of staining
On the basis of staining property
a. Positive staining: Gram staining, AFB staining, H/E
staining etc.
b. Negative staining: Stains the background material
e.g. India ink to demonstrate the capsulated yeast cells
in CSF

6. On the basis of number of stains used
a. Simple staining: e.g. Methylene blue ,Wayson’s stain .
b. Differential staining: e.g. Gram staining , AFB
staining .

7. Introduction
 Very useful stain in diagnostic Microbiology
 Several modifications after discovery
 No technology developed to substitute Gram’s stain till the
date
 Very easy to perform within short time period
 Helps in rapid diagnosis of several microbial diseases
 Guides physician to start appropriate antimicrobials
providing evidence of Gram positive and Gram negative
bacteria from clinical specimen

8. Solutions used
I. Primary stain- Crystal violet
II. Mordant- Gram’s Iodine
III. Decolorizer- Acetone or alcohol or mixture of
acetone alcohol
IV. Counter stain- Neutral red or Safranine

9. Importance of Gram’s stain
 Important test for the rapid presumptive diagnosis of
infectious agent
 To classify the bacteria as a Gram positive or Gram negative
 To study the morphology of bacteria
 To study the arrangement of bacteria
 To find out the evidence of capsule
 To find out the evidence of spore
 To find out the evidence of pus cells
 To find out the evidence of epithelial cells
 To find out the evidence of Yeast cells

11. Difference betweenGrampositive and Gramnegative bacteria
Peptidoglycan
Lipopolysaccharid
e
Techoic acid/
Techuronic acid
PH of protoplasm
Gram positive
Thick layer
Thin layer
Present
2-3
Gram negative
Thin layer
Thick layer
Absent
4-5

13. Principle of Gram’s stain
Three hypothesis
a. Cell wall related
b. PH related
c. Magnesium ribonuclease related

14. PRINCIPLE
Cell wall permeability hypothesis:
 Gram positive cell wall has thicker peptidoglycan layer as
compared to Gram negative cell wall .
 Gram negative cell wall contains a thick layer of
lipopolysaccharide in their cell wall which is absent in Gram
positive bacteria .
 During Gram staining a dye iodine complex (CVI complex)
or lake is formed within the cell wall after staining with crystal
violet and on subsequent treatment with iodine .This complex
is insoluble in water but soluble in alcohol.
 Now when we add decolourizer(Acetone alcohol ),the dye
iodine complex is trapped inside the thick peptidoglycan layer
of Gram positive bacteria so they are not decolorized .

15. Cont …..
 But outer lipopolysacharide layer of Gram negative cell
wall is easily dissolved by the decolourizer due to which
dye iodine complex is easily washed away from Gram
negative cell.

16. Principle
Presence of magnesium ribonucleate :
 Magnesium ribonucleate present in Gram positive
bacteria has affinity for basic dyes. As a result Gram
positive bacteria take the color of crystal violet .
 It has also been proved that Gram positive bacteria
become Gram negative upon removing this material.

17. Principle
 pH hypothesis :
Cytoplasmic pH of Gram positive bacteria is more
acidic (2-3) as compared to Gram negative bacteria(4-
5) and since the primary stain used(crystal violet ) is
basic in nature it develops more affinity with the Gram
positive cytoplasm .
 The difference in pH is mostly due to presence of
Teichoic acid in the cytoplasm of gram positive cell,
which is absent in Gram negative cell .

18. Reagentpreparation
 Crystal violet –Primary stain (0.5 -2%)
Crystal violet – 20gm
Ammonium oxalate -9gm
Absolute ethanol or methanol -95ml
Distilled water –Upto to 1 liter.
• Lugol’s iodine solution –Mordant
Potassium iodide - 20gm
Iodine - 10gm
Distilled water -Upto 1 liter
Note : Lugol's Iodine = 20g Potassium Iodide + 10g Iodine dissolved in
1L H2O
Gram's Iodine = 6.7g potassium iodide + 3.3g Iodine dissolved
in 1L H2O

19. Cont…..
 Acetone alcohol -Decolourizer
Acetone -500ml
Ethanol or Methanol absolute -475ml
Distilled water - 25ml
 Neutral red – Counter stain (0.1%)
1:10 diluted carbol fuschin or saffranin (0.5%) can also
be used .The use of dilute carbol fuschin is
recommended for staining Yersinia Campylobacter,
Hemophilus and Vibrio species .
 Precaution :Ethanol and acetone being flammable , has
to be used away from flame .

20. Storage and stability
 Reagents should be stored in brown glass bottles at
room temperature .
 Crystal violet is stable for 1 year .
 Lugol’s iodine : 6 months .
 Acetone alcohol : Indefinitely .
 Neutral red : 1 year .

21. The GramStain Procedure
•Step 1 - Prepare a Smear
Watch what happens to the “Bacteria” at each step
“Bacteria”
Slide has to be perfectly clean and smear has to be about
1.5 cm in diameter
Allow to air dry. Heat fix by gently warming
above a flame or other heat source.
Note :For detection of Gonococci and Meningococci
:methanol fix for 5 minutes to avoid damaging of pus
cellls .

22. The Gram Stain Procedure
•Step 2 - Apply the Primary Stain
Flood the Smear with Crystal Violet
Allow to stand 1 min
Rinse with water to remove excess stain

23. The Gram Stain Procedure
•Step 3 - Apply the Mordant
Flood the Smear with Iodine solution
Allow to stand 1 min

24. The Gram Stain Procedure
•Step 4 - Rinse
Rinse with water to remove excess Iodine

25. The Gram Stain Procedure
•Step 5 - Decolorize
Cover the smear with 70 % Acetone Alcohol for 30 sec
The effluent should appear pale or clear

26. The Gram Stain Procedure
•Step 6 - Rinse
Rinse with water to remove excess alcohol

27. The Gram Stain Procedure
•Step 7 - Counterstain
Flood the slide with Safranin solution
Let stand 1 minute.

28. The Gram Stain
•Step 8 - Rinse, Dry and Observe
Gram-Positive Gram-Negative
Rinse with water to remove excess stain
Blot dry
Observe under Oil Immersion

29. Examples of Gram Stains
Gram Positive Rods
and Cocci
Gram Negative Rods
and Cocci

30. RESULTS
Gram positive bacteria………..Dark purple
Yeast cells……………………...Dark purple
Gram negative bacteria…………Pale to dark red
Epithelial cells…………………..Pale red

31. Reporting of Gramsmears
The report should include the following information:
- Number 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.
- Whether the organisms are intracellular.
- Presence and number of pus cells.
- Presence of yeast cell and epithelial cells

32. Result and interpretation
a. Evaluate the general nature of the smear under
low-power magnification (10X).
Determine if smear has been properly decolorized.
Determine if thickness of smear is appropriate.
b. Examine under low power for the evidence of
inflammation
Relative amount of WBCs
Relative amount of epithelial cell
c. Location and arrangement of microorganism

33. Variation in Gram reaction.
Gram positive organism may lose their ability to retain crystal violet and stain Gram
negative for the following reasons:
Cell wall damage due to antibiotic therapy or excessive heat fixation of the
smear.
Over de-colorization of the smear
Use of an iodine solution which is too old
Smear that has been prepared from an old culture
Gram negative organisms may not be fully decolorized and appear as gram positive
when a smear is too thick.

34. Recording observations
a. Record relative amount of observed cells and
microorganism
 Numerical
1+ (< 1 per oil immersion field, 100x)
2+ (1 per oil immersion field)
3+ (2 to 10 per oil immersion field)
4+ (predominant or >10 per oil immersion field)

35.  Descriptive
Rare (<1 per oil immersion field)
Few (1 to 5 per oil immersion field)
Moderate (5 to 10 per oil immersion field)
Many (>10 per oil immersion field)

36. Quality control
 Check appearances of chemicals daily.
 Daily and when a new lot of stain is used, prepare a
smear of E. coli, S. epidermidis or S. aureus, fix and
stain.
 Use a set of reference slide.
 To avoid the errors resulting from dirty slides use the
slides previously dipped in 95% Ethanol and soak prior
to use.

37. Limitations of Gram stain
 The number of microorganism required is
very high, for visualizing with Gram stain
(>10000 organisms/ml)
 Can not give good results under following
conditions:
a. Cell wall damage due to antibiotic
therapy or over heat fixation of smear
b. Use of old cultures
c. Uneven thickness of smear
d. Use of old iodine solution
e. Over decolorization of smear

38. Gram stain modifications and uses
Stain and use Original Hucker’s
(1921)
Kopeloff and
Beerman’s
Primary stain Gentian violet Crystal violet Alkaline crystal
violet
Mordant Lugol’s iodine Gram’s iodine Kopeloff’s iodine
Decolourizer Absolute
alcohol
Acetone
alcohol
3:7 acetone alcohol
Counter stain Bismark
brown
Safranin Kopeloff’s safranin
Use General
bacteriology
Anaerobes , For
diagnosis of
bacterial vaginosis .

39. Gram stain modifications and uses
Stain and use Jensen’s Preston and
Morrell’s(1962
)
Brown and
Brenn
Weigert’s
Primary stain 0.5%methyl
violet
Ammonium
oxalate-crystal
violet
1%Crystal
violet
Carbol gentian
violet
Mordant 1%Lugol’s
iodine
1%Lugol’s
iodine
Lugol’s Iodine Gram’s iodine
Decolourizer Absolute
alcohol
Iodine –
acetone
Acetone Aniline xylene
Counter stain 0.1% Neutral
red
Dilute carbol
fuschin
0.25%Basic
fuschin
Carminic acid
and potassium
alum in water .
Use For Gonococci
and
Meningococci
Bacteroides
spp,Fusobacte
rium species ,
For
Demonstrating
Bacteria in
tissue .
Pneumocyst
,Nocardia .

40. Applications of Gram’s Staining
 Used to guide initial therapy until definitive
identification of microorganism obtained.
 Morphology of stained bacteria can sometimes be
diagnostic. For example Gram negative intracellular
diplococci in urethral pus provides a presumptive
diagnosis of Gonorrhea .
 Sometimes specimens may show organisms under
microscope but appear sterile in culture media .In
these cases Gram stain is the only clue to the nature
,variety and relative proportion of infecting organism
.
 Aids in interpretation of culture reports .