‫‪PREPARED BY‬‬

‫عالء عطشان عجيل فاطمه صادق عبد االمير‬

‫لبنى هارون عبد النبي‬ ‫علي عباس‬
‫محمد شاكر عبد الواحد‬ ‫علي ماجد محمد‬
‫علي مكي عبد الحسن محمد عبد الستار‬
‫محمود سعد‬ ‫علياء فاضل خلف‬

‫‪Supervised by‬‬

‫‪Date:2021\11\25‬‬ ‫‪Dr: zainab safaa‬‬

‫‪0‬‬
Introduction
Staining is a technique used to enhance contrast in samples, generally at the
microscopic level. Stains and dyes are frequently used in histology (microscopic study
of biological tissues) and in the medical fields of histopathology, hematology, and
cytopathology that focus on the study and diagnoses of disease at a microscopic level.
Stains may be used to define biological tissues (highlighting, for example, muscle fibers
or connective tissue), cell populations (classifying different blood cells), or organelles
within individual cells. A stained histologic specimen, sandwiched between a glass
microscope.

In biochemistry it involves adding a class-specific (DNA, proteins, lipids,

carbohydrates) dye to à substrate to qualify or quantify the presence of a specific
compound. Staining and fluorescent tagging can serve similar purposes.
Biological staining is also used to mark cells in flow cytometry, and to flag proteins
or nucleic acids in gel electrophoresis. Light microscopes are used for viewing stained
samples at high magnification, typically using bright-field or epi-fluorescence
illumination. The method is named after its inventor, the Danish scientist Hans
Christian Gram (18531 938), who developed the technique while working with Carl
Friedländer in the morgue in 1884 off the city hospital in Berlin in Gram devised I,
this technique is not for the purpose of distinguishing one type of bacterium from
another but to make bacteria more visible in stained sections of issue.
He published his method in 1 884, and included in his short report lung tissue. the
observation that the typhus bacillus did not retain the stain. Gram stain or Gram
staining, also called Gram's method, is a method of staining used to classify bacterial
species into two large group gram-positive bacteria and gram-negative bacteria.
Gram-positive cells have a thick layer of peptidoglycan in the cell wall that retains the
primary stain, crystal violet. Gram-negative cells have a thinner peptidoglycan layer
that allows the crystal/violet to wash out on addition of ethanol. They are stained pink
or red by the counterstain, commonly safranin or fuchsine.

1
Types of dyes or reagents
Safranin:
Safranin is a biological stain used in histology and cytology. Safranin is used as a
counterstain in some staining protocols, coloring all cell nuclei red. This is the classic
counterstain in a Gram stain. It can also be used for the detection of cartilage, mucin
and mast cell granules. Safranin typically has the chemical structure shown at right.
There is also trimethyl safranin, which has an added methyl group in the ortho-
position of the lower ring. Both compounds behave essentially identically in biological
staining applications, and most manufacturers of safranin do not distinguish between
the two. Commercial safranin preparations often contain a blend of both types.
Safranin is also used as redox indicator in analytical chemistry.

Gram's iodine:
is used in Gram staining procedure to differentiate gram positive and gram-negative
organisms. Gram's lodine acts as a mordant that causes the crystal violet to penetrate
and adhere to the gram-positive organisms. The Gram stain is a differential staining
technique most widely applied in all microbiology disciplines laboratories. It is one of
the most important criteria in any identification scheme for all types of bacterial
isolates. Different mechanisms have been proposed to explain the gram reaction.
There are many physiological differences between gram-positive and gram-negative
cell walls

Decolorized or Ethyl alcohol:

is a nonpolar solvent, and thus penetrates the cell walls of Gram negative cells more
readily and removes the crystal violet-iodine complex. However, caution must be used
since applying the decolorizer too long will remove dye complexes from the Gram-
positive cells as well.
Crystal violet staining solution was Crystal violet staining solution is prepared in the
same way of liquid a used in Gram stain. Take a small quantity of culture and mix
with physiological saline to prepare a smear. Stain the smear with crystal violet
solution.

2
Material and tools

1-Loop
2-Benzene burner
3-Slides
4-Filter paper
5-Dropper
6-Distilled water
7-culture medium containing bacterial growth

How to stain bacterial

The method consists of three steps:

Coloring-removing coloring- then the opposite coloring

1- In the first stage, the coloring is done with Gentian violet (or known as Crystal
violet) added to Phenol.
(Historically known as Carbol). Gram-positive and gram-negative bacteria color at
that time. Then the bacteria are treated with Lugol's solution, and then compounds of
coloring materials are formed.

2- In the second step- which is the task- the reaction of the bacteria varies, after
treatment with alcohol-ethanol (96%)-the negative bacteria lose their acquired color
initially, while positive ones do not give up their dark blue color that you acquired.
This difference is due to the structure of their cell wall. Negative bacteria have a thin
layer of murein, but above it a phospholipid layer.

3
Whereas, the cation has a thick, multi-layered murine sheath between which Lugol's
solution accumulates. Here, the effect of ethanol is drying. as the color aggregates
remain available in the cell wall.
This stage-i.e., alcohol therapy-requires some experience to establish a definitive
diagnosis

3- The third step includes staining the bacteria

with a buffing solution-ie. of low concentration - from the auctioned fuchsin to phenol.
(Therefore, the red color is produced in Gram-negative bacteria according to the Gram
staining)

There are also other ways to find out the behavior of bacteria on a gram basis, such as
the KOH test Method Aseptically a small sample of the culture is spread over a slide
surface. This is then allowed to air dry.
The next step is heat fixation to help the cells adhere to the slide surface. The smear is
now ready for staining.

4
Preparation of bacterial smear

1-Put one drop of distilled water in the center of clean, dry

slide Sterile wire loop with burner flame, then take a small
amount from bacterial growth.
2-Emulsify the growth with distilled water drop and spread it
on slide.
3-Leave the smear to dry.
4-Fixed the smear by flame by passing the slide quickly 2-3
times on flame.
5-The smear ready now for staining.

Bacterial Staining

1. put the slide on the staining rack.

2. flood it with crystal violet stain, leave it for 60 seconds.
3. Wash the slide carefully by washing bottle.
4. Hood the slide with iodine stain, after 60 seconds wash it by distilled water.
5. flood the slide with decolorizer reagent for 30-35 seconds. then wash it by
distilled water.
6. flood the slide with safranin for 60 seconds, then wash it by distilled water.
7. dry the slide with filter paper.
8. examine your slide under microscope, then by oil Immersion

5
Discussion

The Gram stain is the most widely used staining procedure in bacteriology. It is called
a differential stain since it differentiates between Gram-positive and Gram-negative
bacteria. Bacteria that stain purple with the Gram staining procedure are termed Gram-
positive; those that stain pink are said to be Gram negative. The terms positive and
negative have nothing to do with electrical charge, but simply designate two distinct
morphological groups of b Gram-positive and Gram-negative bacteria stain differently
because fundamental differences in the structure of their cell walls. The bacteria serve
to give the organism its size and shape as well as to prevent osmotic the material in the
bacterial cell wall which confers rigidity is peptidoglycan.
With the current theory behind Gram staining, it is though that in Gram-positive
bacteria, the crystal violet and iodine combine to form a larger molecule that
precipitates out within the cell. The alcohol/acetone mixture then causes dehydration
of the multilayered peptidoglycan, thus decreasing the space between the molecules
and causing the cell wall to trap the crystal violet-iodine complex within the cell. In
the
case of Gram-negative bacteria, the alcohol/ acetone mixture, being a lipid solvent,
dissolves the outer membrane of the cell wall and may also damage the cytoplasmic
membrane to which the peptidoglycan is attached. The few layers of peptidoglycan are
unable to retain the crystal violet iodine. complex and the cell is decolorized. It is
important to note that Gram-positivity (the ability to retain the purple crystal violet
iodine complex) is not an all-or-nothing phenomenon but a matter of degree. There are
several factors that could result in a Gram-positive organism staining Gram-
negatively:
1. The method and techniques used. Overheating during heat fixation, over
decolorization with alcohol, and even too much washing with water between steps
may result in Gram-positive bacteria losing the crystal violet-iodine complex.
2. The age of the culture. Cultures more than 24 hours old may lose their ability to
retain the crystal violet-iodine complex.
3. The organism itself. Some Gram-positive bacteria are more able to retain the
crystal violet-iodine complex than others.

6
Sources

❖ Charras GT, Coughlin M, Mitchison TJ, Mahadevan L (Mar 2008). "Life and
times of a cellular bleb". Biophys.

❖ Charras GT, Hu CK, Coughlin M, Mitchison TJ (Nov 2006). "Reassembly of

contractile actin cortex in cell blebs".

❖ Dal J, Sheetz MP (Dec 1999). "Membrane tether formation from blebbing

cells". Biophys. J. 77 (6): 3363-70.

❖ Drug Stops Motor Protein, Shines Light on Cell Division - FOCUS March 21,
2003. Retrieved April 8, 2008.