Sigma-Aldrich Gram-Color Manual

1.11885.0001
Microscopy
Gram-Color
stain set for the Gram staining method
For professional use only
In vitro Diagnostic Medical Device
Intended purpose
This "Gram-Color - stain set for the Gram staining method" is used for
human-medical cell diagnosis and serves the purpose of the bacteriologi-
cal investigation of sample material of human origin. It is a ready-to-use
staining kit that when used together with other in vitro diagnostic products
from our portfolio makes bacterial target structures evaluable for diagnos-
tic purposes (Gram-positive or Gram-negative bacteria) by fixing, stain-
ing, counterstaining, mounting in bacteriological specimen materials, e. g.
smears of body fluids.
The Gram-Color solutions are modified and designed in such a way that
staining can be carried out in staining cells, on the staining rack, and also in
automated staining systems.
Unstained structures are relatively low in contrast and are extremely diffi-
cult to distinguish under the light microscope. The images created using the
staining solutions help the authorized and qualified investigator to better
define the form and structure in such cases. Further tests must be carried
out according to recognized, valid methods to reach a definitive diagnosis.
Principle
In bacteriology, the Gram staining allows a fast differentiation of bacteria in
Gram-positive and Gram-negative.
The mureine structure of the bacteria wall is the basis of the color affinity.
In the first step, bacteria will be stained with crystal violet, an aniline dye.
After the treatment with iodine solution (Lugol's solution), a dye-iodine
complex will form. During the decolorizing step, this complex stays in the
multi-layer mureine structure of the cell wall of Gram-positive bacteria -
they will appear blue-violet.
Gram-negative bacteria, by contrast, have a cell wall consisting of a single-
layered murein structure, and correspondingly re-release the staining dye
with the decoloring solution. Gram-negative bacteria will be counterstained
with safranine solution and will then appear pink to red.
Sample material
Smears of bacteriological material that have been air-dried and heat-fixed
like sputum, smears from fine needle aspiration biopsies (FNAB), rinses,
imprints, effusions, pus, exudates, liquid and solid cultures
Reagents
Cat. No. 1.11885.0001
Gram-Color
stain set for the Gram staining method
Package components:
The staining kit contains
Reagent 1: Crystal violet solution
Reagent 2: Lugol's solution, stabilized
Reagent 3 = 4: Decolorizing solution
Reagent 5: Safranine solution
Single reagents
Instead of the staining kit 1.11885.0001, the following combination of
reagents can be used:
Cat. No. 1.09218 Gram's crystal violet solution
for the Gram staining method
Cat. No. 1.00567 Lugol's solution stabilized with PVP
for the Gram staining method
Cat. No. 1.09261 Lugol's solution (diluted iodine-potassium
iodide solution)
for the Gram staining method
Cat. No. 1.10218 Gram's decolorizing solution
for the Gram staining method
Cat. No. 1.09217 Gram's safranine solution
for the Gram staining method
Sample preparation
The sampling must be performed by qualified personnel.
Apply the specimen material to a clean and grease-free slide using an an-
nealed loop. Then smear the material either directly onto the slide or first
mix with 1 - 2 drops of physiological saline solution (Ringer's solution).
Air-dry and then heat-fix by slowly drawing the slide (smear side facing up)
through the upper part of the Bunsen-burner flame for three times. Subse-
quently, allow to cool and stain.
The air-dried smears must be heat-fixed very carefully. This prevents the
risk of infections and reduces the dissolution of specimen material and thus,
the contamination of solutions and other slides.
All samples must be treated using state-of-the-art technology.
All samples must be clearly labeled.
Suitable instruments must be used for taking samples and their prepara-
tion. Follow the manufacturer's instructions for application / use.
When using the corresponding auxiliary reagents, the corresponding in-
structions for use must be observed.
Reagent preparation
The reagents of the Gram-Color - stain set for the Gram staining method
used for staining are ready-to-use, dilution of the solutions (except for
reagent 1 for staining in the cell - see "Procedure") is not necessary.
Procedure
Staining in the staining cell
It is recommended to dilute the reagent 1 (crystal violet solution) 1:3 with
distilled water, if the immersion method is used.
The slides must be immersed and moved about in the solutions, simple im-
mersion alone yields inadequate staining results.
The slides should be allowed to drip off well after the individual staining steps
as a measure to avoid any unnecessary cross-contamination of solutions.
The stated times should be adhered to in order to guarantee an optimal
staining result.
Slide with fixed smear
Reagent 1 (crystal violet solution)
Running tap water
Reagent 2 (Lugol's solution, stabilized)*
Running tap water
Reagent 3 or 4 (decolorizing solution)**
Running tap water
Reagent 5 (safranine solution)
Running tap water
Air-dry (e. g. over night or at 50 °C in the drying cabinet)
* filter reagent 2 (Lugol's solution, stabilized) after 3 runs
** discard reagent 3 or 4 (decolorizing solution) after 5 runs
Staining on the staining rack
Slide with fixed smear
Reagent 1 (crystal violet
solution)
500 ml
Reagent 2 (Lugol's solution,
500 ml
stabilized)
each 500 ml
Reagent 2 (Lugol's solution,
500 ml
stabilized)
Distilled water
Reagent 3 or 4 (decolorizing
solution)
500 ml, 2.5 l
1 l, 2.5 l
Distilled water
1 l, 2.5 l Reagent 5 (safranine solu-
tion)
Distilled water
500 ml, 2.5 l
Air-dry (e. g. over night or at 50 °C in the drying cabinet)
500 ml, 2.5 l
Covering with non-aqueous mounting media (e. g. Neo-Mount
and a cover glass is recommended for the storage of bacteriological speci-
mens for several months. For this purpose, the stained specimens must be
dried very well.
The use of immersion oil is recommended for the analysis of stained slides
with a microscopic magnification >40x.
www.sigmaaldrich.com
1:30 min
30 sec
20 sec
5 - 10 sec
30 sec
cover completely and leave
to react
rinse briefly
cover completely and leave
to react
wash carefully
carefully swirl the slides un-
til no further clouds of dye
are produced and the smear
takes on a grey-blue color
wash carefully
cover completely and leave
to react
wash carefully
or Entellan
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EN
3 min
1 min
1 min
1 min
1 min
5 sec
10 - 15 sec
5 sec
1 min
5 sec
)
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