DEPARTMENT OF MICROBIOLOGY

TIRUNELVELI MEDICAL COLLEGE TIRUNELVELI.

GRAM STAINING

1. What is stain or dye?

Stain or dye is the synthetic chemical which is derived from
nitrobenzene or aniline
Stains are used in microbiology to increase the contrast between
microorganism or its parts and the background, so that it can be easily
visible.
2. What are the types of Staining?
 Simple Staining-Only one stain is used to stain the cells.
 Negative Staining
 Differential Staining-More than one stain is used for staining.
3. What are Positive and Negative staining?
 Positive Staining-A dye that will be absorbed by the cells or
organisms being observed,addingcolor to them to make them stand
out against the background.Bacteria is stained eg.,Gram staining.
 Negative Staining-A dye which is absorbed by the background but
not by the cells or organisms in the specimen. Background is
stained.eg., Indian Ink,Capsular Nigrosine staining.
4. Name three Acidic and Basic stains( dyes)?
o Acidic stain-Acid fuchsin,Eosin,Picric acid,Rose Bengal,Congo
Red.
 Basic stain-Crystal violet,malachitegreen,safranin,Carbol fuschin.
5. What is differential staining?
When two stains are used to impart different colors to different
bacteria or bacterial structures it is called differential staining.
 Eg.Gramsstaining ,Acid Fast staining

6. Who invented Gram stain?

Danish bacteriologist
Hans Christian Gram in 1884

7. What is the principle of Gram stain?

1.PH theory(Acidprotoplasmic theory)-
Cytoplasm of gram positive bacteria is more acidic,hence retain
basic dye(crystal violet) for longer time.
Iodine serves as a mordant. It combines with the primary stain to form a
dye iodine complex which gets retained inside the cell.

2.Cell wall theory-

Gram negative cell wall:
Gram negative cell wall has a thin peptidoglycan layer which is
not tightly cross linked.
Cell wall of gram negative bacteria gets disrupted easily by the
action of acetone or alcohol due to presence of lipopolysaccharide layer
allowing the primary stain to come out of the cytoplasm.

Gram Positive Cell Wall:

Gram positive cell wall has a thick peptidoglycan layer with tight
cross linkages.
Peptidoglycan layer acts as a permeability barrier preventing loss
of crystal violet.
 3.Porin theory:
Increasesdlipid content in gram negative cell wall gets dissolved
following decolorisation leading to formation of larger pores through
which the dye iodine complex escape.
Less Lipid in gram positive bacterial cell wall, smaller pores are
formed which do not allow the dye iodine complex to escape.

4.MagnesiumRibonucleotide theory:
 In Gram positive bacteria,dye iodine complex combines with
theMg-RNA component of cell wall to form the larger ‘Mg-RNA -
dye-iodine complex,which gets retained inside the cell.

8. Differences between Gram positive and Gram negative cell wall?

Properties Gram positive Gram Negative

Thickness Thicker Thinner
Teichoic acid Present Absent
Lipids Absent/Scanty Present
Aromatic and Absent Present
Sulphur
containing amino
acids
Peptidoglycan 50-90% Of the 5-10% Of the
dry weight of dry weight of
the cell wall. the cell wall
 9. What are Gram variable Bacteria?
 Gram positive bacteria that have lost their cell wall integrity
because of antibiotic treatment,old age or action of autolytic
enzymes.
 These changes allow crystal violet to come out of the cell wall
during process of decolourising resulting in some cells staining
pink and others staining purple.

10.When will Gram positive organism appear as Gram negative (gram

variable)?
 Overdecolorisation of the smear
 Smear prepared from old cultures
 Use of iodine solution which is too old
 Cell wall damage due to excessive heat fixation of the smear.
 Action of autolytic enzymes
 Following antibiotic therapy

11.What is the procedure of Gram staining?

STEP 1:Primary staining
 Flood methyl violet over smear
 Wait for 1 minute
 Wash the smear with water.
 Stains all the bacteria violet in color
STEP 2: Fixation of the primary stain or Mordanting
 Flood Gram’s iodineover smear
 Wait for 1 minute
 Wash the smear with water.
 Grams Iodine contains
Iodine -1gm
Potassium iodide 2gm
Distilled water 300ml
Iodine serves as a mordant.
Iodine combines with the primary stain to form a dye iodine complex
which gets retained inside the cell.

STEP 3:Decolorisation
 Flood Acetone over the smear
 Immediately wash (1-2 sec)the smear with water.

Decolorizer–
 Removes the primary stain from the bacteria
 Gram negative bacteria are decolourised.
 While gram positive bacteria resist the decolouriser and retain the
primary stain.

STEP 4:Counterstaining
Flood the smear with dilute carbolfuchsin
Wait for 1 minute
Wash the smear with water
Air dry –view under oil immersion.
What is the crucial step in gram staining?
Decolorisation step - as over decolorisation or under decolorisation may
facilitate gram positive to appear as gram negative and vice versa
12.What are the primary stains of gram staining?
 Crystal violet
  Gentian violet
 Methyl violet.

13.What are mordants?

 Mordants are substances used in staining treatment which penetrate
into a bacterial cell .
 They dehydrate cell and shrink the pores in cell wall come closer
and help to retain the original dye within the cell.
14.What are the decolourisers used?
 Acetone (1-2 seconds )
 Ethyl alcohol& methanol -20-30 seconds ( highly flammable )
 Acetone alcohol 10 seconds
 Iodine acetone.
15.What are the counterstains used?
 Safranin
 Dilute carbolfuschin
 Neutral red
16.What are the microscopic adjustments for viewing Gram stained smear ?
 Smear is examined under oil immersion objective.
 Open the diaphragm
 Plane mirror
 Raise the condenser
17.Name few Gram positive cocci?
1. Staphylococcus aureus
2. Streptococcus species
3. Enterococcus faecalis
18.Name few Gram positive bacilli?
1. Clostridum
2. Corynebacterium
3. Bacillus
4. Actinomyces
5. Nocardia

19.Name few Gram negative cocci?

1. Meningococci
2. Gonococci
3. Veillonella

20.Name few Gram negative bacilli?

1. Escherichia coli
2. Klebsiella species
3. Pseudomonas aeruginosa

21.Name organisms which lack cell wall?

1. Mycoplasma
2. Ureaplasma
3. Spiroplasma
4. Anaeroplasma
 22.What are L forms?
1. Lforms are the cell wall deficient bacteria.(L-lister)
Discovered by EmmyKlienberger,while studying
Streptobacillusmoniliformis and named them after Lister institute
in London where she was working.
When bacteria loose cell wall,they become spherical irrespective of
original shape.
2. Bacteria lose the cell wall in the presence of penicillin, a
mechanism of resistance shown by the bacteria against penicillin.
3. Such L forms are maintained only in presence of penicillin.
4. They are capable of dividing, but can revert back to the original
morphology once penicillin is removed.

23. What are Protoplasts and Spheroplasts:

 Protoplasts:They are gram positive bacteria whose cell wall is
entirely removed.
 Spheroplasts:They are derived from gram negative bacteria whose
cell wall is partially removed.

24.Name few motile bacteria?

Types of motility Bacteria
Tumbling motility Listeria
Darting motility Vibrio cholera,campylobacter
Swarming on agar plate Proteus,clostridium tetani
Gliding Mycoplasma
Corkscrew Spirochetes
Stately Clostridium

25.Give examples for

 Arrangement of bacteria:
a. Gram positive cocci :
i. Clusters-Staphylococcus
ii. Chains-Streptococcus
iii. Tetrads-Micrococcus
iv. Octate-Sarcina
v. Pair,-Pneumococcus
vi. Pair or short chains-Entrococcus

Shapes of bacteria
b. Gram negative cocci:
i. Pairs,lens shaped-Meningococcus
ii. Pairs,kidney shaped-Gonococcus
iii. lanceolate shaped- Pneumococcus
Appearance of bacteria :
Gram positive bacilli:
i.Chains(bamboo stick appearance)-Bacillus anthracis
ii.Chinese letter or Cuneiform pattern-Corynebacterium
diphtheria
Gram negative bacilli:
i.Pleomorphic(variation in size and shape of bacteria )-
Haemophilus,Proteus
ii.Thumb print appearance-Bordetella Pertussis
iii.Comma shaped-Vibrio cholera.

26.What is the use of oil in oil immersion?

 a. Oil has a higher refractive index than air;hence use of oil
enhances the resolution power of a microscope.
27.How do you fix the smear?
a. Heat fixation-It is done by gently flame heating andair dried film
used for bacterial smears.
b. Methanol fixation.
28.How do you clean the glass slide?
a. It should be clean with soft tissue paper
b. It should be grease free.
29.Uses of Gram staining?
a. To differentiate bacteria into gram positive and gram negative.
b. For identification of bacteria
c. To start empirical treatment.
d. To procede with the appropriate biochemical tests .

30.What are the modification of Gram staining?

Method Modification Use
Kopeloff and Beerman’s Primary stain-methyl Gram staining
modification violet Counter stain-basic
fuchsin.

Jensen’s modification Decoloriser-absolute Meningococci and

alcohol,Counter stain- Gonococci.
neutral red.

Weigert’s modification- Decoloriser-aniline xylol. For tissue sections.

staining
Preston and Morrell’s Decoloriser-iodine Grams staining
modification – acetone.
31.What are the other Structures identified by Gram staining other than
bacteria?
e. Candida -- fungus
f. Cryptococcus --fungus
g. Pus cell
h. Epithelial cell.

32. What is direct gram staining ?

When gram staining is done directly on the specimen
eg .sputum ,CSF,pleural fluid .

33. Name some Special staining ?

Alberts staining – Corynebacteriumdiphtheria .
Spore staining - Schaffer fulton method
Flagellar staining – RYU method