GROUP 5- Activity Number 8 (Gram Stain Method)

Enrichment:

1. Discuss the various theories on the Gram stain.

Staining is an auxiliary technique used in microscopic techniques used to enhance the clarity of
the microscopic image.Stains and dyes are widely used in the scientific field to highlight the structure of
the biological specimens, cells, tissues etc.

The most widely used staining procedure in microbiology is the Gram stain, discovered by the
Danish scientist and physician Hans Christian Joachim Gram in 1884. Gram staining is a differential
staining technique that differentiates bacteria into two groups: gram-positives and gram-negatives. The
procedure is based on the ability of microorganisms to retain color of the stains used during the gram
stain reaction. Gram-negative bacteria are decolorized by the alcohol, losing the color of the primary
stain, purple. Gram-positive bacteria are not decolorized by alcohol and will remain as purple. After
decolorization step, a counterstain is used to impart a pink color to the decolorized gram-negative
organisms.
Details of the chemical mechanism of the Gram stain were determined in 1983 (Davies et
al.,1983 and Beveridge and Davies, 1983). In aqueous solutions crystal violet dissociates into CV+ and
Cl ions that penetrate through the wall and membrane of both gram-positive and gram-negative cells.
The CV+ interacts with negatively charged components of bacterial cells, staining the cells purple.
When added, iodine (I- or I3-) interacts with CV+ to form large CVI complexes within the cytoplasm
and outer layers of the cell. The decolorizing agent, (ethanol or an ethanol and acetone solution),
interacts with the lipids of the membranes of both gram-positive and gram-negative Bacteria. The outer
membrane of the gram-negative cell is lost from the cell, leaving the peptidoglycan layer exposed.
Gram-negative cells have thin layers of peptidoglycan, one to three layers deep with a slightly different
structure than the peptidoglycan of gram-positive cells (Dmitriev, 2004).With ethanol treatment, gram-
negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell. The
highly cross-linked and multi-layered peptidoglycan of the gram-positive cell is dehydrated by the
addition of ethanol. The multi-layered nature of the peptidoglycan along with the dehydration from the
ethanol treatment traps the large CV-I complexes within the cell. After decolorization, the gram-positive
cell remains purple in color, whereas the gram-negative cell loses the purple color and is only revealed
when the counterstain, the positively charged dye safranin, is added. At the completion of the Gram
stain the gram-positive cell is purple and the gram-negative cell is pink to red.

2. Tabulate the main differences between gram positive from gram negative bacteria.

Gram-negative Gram-positive Bacteria

Bacteria
Gram reaction: Can be decolourized Retain crystal violet dye
to accept counter and stain dark violet or
stain (Safranin or purple, they remain
Fuchsine); stain red coloured blue or purple
or pink, they don't with gram stain when
retain the Gram stain washed with absolute
when washed
 with absolute alcohol and water.
alcohol and acetone.
Peptidoglycan Thin (single-layered) Thick (multilayered)
layer:
Teichoic acids: Absent Present in many

Periplasmic space: present Absent

Outer membrane: Present Absent

Lipopolysaccharide High Virtually none

(LPS) content:
Lipid and High (due to Low (acid-
lipoprotein presence of outer fast bacteria have lipids
content: membrane) linked to peptidoglycan)

Flagellar structure: 4 rings in basal body 2 rings in basal body

Toxinsproduced: Primarily Endotoxins Primarily Exotoxins

Resistance to High Low

physical
disruption:
Inhibition by basic High Low
dyes:
Susceptibility to High Low
anionic detergents:
Resistance to High Low
sodium azide:
Resistance to High Low
drying:
Cell wall The cell wall is 70- The cell wall is 100-120
composition: 120 Armstrong thick Armstrong thick, single
two layered.The lipid layered. The Lipid content
content is 20-30% of the cell wall is low ,
(High), whereas whereas Murein content is
Murein content is 70-80% (Higher).
10-20% (Low).
Mesosome: Mesosome is less Mesosome is more
prominent. prominent.

Antibiotic More Resistant More Susceptible

Resistance: to antibiotics. to antibiotics
3. A. Why would you sue a young culture rather than an old culture for the gram stain?

B. Why is it necessary to decolorize only for a few seconds?

Differential extraction of the dye-mordant by the decolorizing agent is the critical step that
distinguishes the bacteria. A counterstain, safranin, is applied in the final step. Cells that have been
decolorized will take up the second basic dye whereas those already stained with the first dye will not.

The mechanism of the differential staining response has not been resolved with certainty. One theory
holds that differences in the cell wall chemical composition account for the staining response. A second
theory maintains that the thicker walls of Gram positive bacteria are dehydrated by the decolorizing
solution and shrink, resulting in the closure of pores in the wall, trapping the dye-mordant within the
cell. The thinner cell wall of Gram negative bacteria would be readily penetrated by the decolorizer.

4. Give specific examples of the following:

a) Gram positive cocci Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus saprophyticus
Streptococcus pyogenes
(Group A, -hemolytic)
Streptococcus agalactiae
(Group B, -hemolytic)
b) Gram negative cocci Neisseria meningitidis
Neisseria gonorrheae
c) Gram positive bacilli Listeria monocytogenes
Bacillus anthracis
Bacillus cereus
Corynebacterium diphtheriae
Clostridium tetani
d) Gram negative bacilli Brucella melitensis
Francisella tularensis
Pasteurella multocida
Yersinia pestis
Yersinia pseudotuberculosis
Yersinia enterocolitica
e) Gram negative curve rod Vibrio cholerae
Vibrio parahaemolyticus
Vibrio vulnificus
Campylobacter jejuni
Campylobacter fetus
Helicobacter pylori
Salmonella enterica
Salmonella typhi
f) Gram negative coccobacili Bordetella pertussis
Haemophilus influenza
Bordetella parapertussis

5. Give the ingredients and preparation of the gram staining reagents.

 (Gephardt et al., 1981 )This is Huckers modification of the Gram Stain method. Gram originally
used Gentian Violet as the primary stain in the Gram stain. Crystal violet is generally used today. In
Huckers method ammonium oxalate is added to prevent precipitation of the dye (McClelland, 2001)
and uses an alcoholic solution of the counterstain. Burkes modification of the Gram Stain adds sodium
bicarbonate to the crystal violet solution. Sodium bicarbonate prevents the acidification of the solution
as iodine oxidizes (McClelland, 2001) and uses an aqueous solution of Safranin for the counterstain
(Gephardt et al., 1981).

The reagents listed below can be made or purchased commercially from biological supply houses

1. Primary Stain: Crystal Violet Staining Reagent.

Solution A for crystal violet staining reagent

Crystal violet (certified 90% dye content), 2g

Ethanol, 95% (vol/vol), 20 ml

Solution B for crystal violet staining reagent

Ammonium oxalate, 0.8 g

Distilled water, 80 ml
Mix A and B to obtain crystal violet staining reagent. Store for 24 h and filter through paper prior to use.

2. Mordant: Gram's Iodine

Iodine, 1.0 g
Potassium iodide, 2.0 g
Distilled water, 300 ml

Grind the iodine and potassium iodide in a mortar and add water slowly with continuous grinding until
the iodine is dissolved. Store in amber bottles.

3. Decolorizing Agent
Ethanol, 95% (vol/vol)

*Alternate Decolorizing Agent

Some professionals prefer an acetone decolorizer while others use a 1:1 acetone and ethanol mixture.
Commercially, a variety of mixtures are available, most using 25 50% acetone with the ethanol. A few
include a small quantity of isopropyl alcohol and/or methanol in the formulation.

Acetone, 50 ml
Ethanol (95%), 50 ml

4. Counterstain: Safranin
Stock solution:
 2.5g Safranin O
100 ml 95% Ethanol
Working Solution:
10 ml Stock Solution
90 ml Distilled water

PROTOCOL (Gephardt et al, 1981, Feedback from ASMCUE participants, ASMCUE , 2005)

1. Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent. Please note
that the quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain results.
2. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
3. Flood slide with the mordant: Gram's iodine. Wait 1 minute.
4. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
5. Flood slide with decolorizing agent. Wait 15 seconds or add drop by drop to slide until decolorizing
agent running from the slide runs clear (see Comments and Tips section).
6. Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute.
7. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then
blot dry with absorbent paper.
8. Observe the results of the staining procedure under oil immersion using a Brightfield microscope. At
the completion of the Gram Stain, gram-negative bacteria will stain pink/red and gram-positive bacteria
will stain blue/purple.

http://www.diffen.com/difference/Gram-negative_Bacteria_vs_Gram-positive_Bacteria

http://amrita.vlab.co.in/?sub=3&brch=73&sim=208&cnt=1

http://www.microbelibrary.org/library/gram-stain/2886-gram-stain-protocols

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