1.11885.

0001
Results
gram-positive microorganisms
gram-negative microorganisms

Microscopy

blue-violet
pink to red

Gram-color
Checking the staining set
The staining set may be checked with gram-positive bacteria (staphylococci)
and gram-negative bacteria (Escherichia coli). Cultures taken from a culture
medium following 18-24 hours of incubation should be used.

staining set for Gram staining

Technical note
The microscope used should meet the requirements of a medical diagnostic
laboratory.

In Vitro Diagnostic Medical Device

Sample preparations

Mode of action

All samples should be treated using state-of-the-art technology.

All samples must be clearly labeled.
Suitable instruments must be used for taking samples and their preparation;
manufacturer instructions for application/use must be followed.

Gram-color is a staining set for staining microorganisms by the

Gram method.

Diagnostics

Aniline dyes in the cell tissue of microorganisms from a red dye-iodine

complex when exposed to iodine.
In gram-positive microorganisms the dye-iodine complex cannot subsequently
be dissolved from the cells with decolourizing agents such as alcohol or
acetone. The cell remains blue-violet.
In gram-negative microorganisms the dye-iodine complex is dissolved and the
cell turns pink to red as a result of counterstaining.

Diagnoses are only to be made by authorized and trained persons.

Valid nomenclatures must be used.
Suitable controls should be conducted with each application in order to avoid
an incorrect result.
Further tests must be selected and implemented according to recognized
methods.

Storage
The staining kit must be stored at +15C to +25C. After the first opening of
the bottle the contents can be used up to the expiry date when stored at
+15C to +25C.

Composition of the staining set::

Cat. No. 1.11885.0001 Gram-color
Solution 1 :
Crystal violet solution
Solution 2:
Lugol's solution stabilized
Solutions 3 + 4: Decolorisation solutions
Solution 5:
Safranin solution

500 ml
500 ml
each 500 ml
500 ml

Single reagents

At temperatures below 15 C a colored precipitate may settle out of the dye

solutions. If precipitation has occurred, place the bottle for 2-3 hours in a water
bath set at about 60 C. Most of the precipitate will re-dissolve. Subsequently
filter the staining solutions through a paper filter.

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
500 ml, 2.5 I
Cat. No. 1.09217 Gram's safranin solution
500 ml, 2.5 I
Cat. No. 1.10218 Gram's decolorisation solution
500 ml, 2.51
Cat. No. 1.00567 Lugol's solution stabilized
1I
Cat. No. 1.09261 Lugol's solution
250 ml, 1 l

Shelf-life

Preparing the smears

Instructions for use

Using an ignited loop, transfer a quantity of specimen, which may be a body

fluid, exsudate, pus, or a liquid or solid culture, on to a degreased slide. Then
distribute the specimen either directly or after adding 1-2 drops of
physiological saline solution. After drying in air, heat-fix the smear by slowly
drawing three times through the upper portion of a Bunsen flame. Leave to
cool and stain.

In order to avoid errors, the staining process must be carried out by an expert.
Only for professional use.
National guidelines for work safety and quality assurance must be followed.
Microscopes equipped according to the standard must be used.
If necessary use a standard centrifuge suitable for medical diagnostic
laboratory.

The bottles must be kept tightly closed at all times.

The solutions must be used by the expiry date stated.

Capacity
The kit is sufficient for up to 250 preparations.

Staining on a staining bench

1.

Completely cover the slide with solution 1 : crystal violet solution.

Stain for 1 min, pour off.
Carefully rinse with solution 2: Lugol's solution stab.
Completely cover the slide with solution 2: Lugol's solution .
stabilized. Allow to act for 1 min.
Carefully rinse with distilled water for about 5 sec.
Swirl the slide for about 10-15 sec in solution 3 or 4;
decolorisation solution. Stop swirling when no more dye is released
and the smear appears greyish-blue.
Carefully rinse with distilled water for about 5 sec.
Completely cover the slide with solution 5: safranin solution. Stain
for 1 min.
Carefully rinse with distilled water for about 5 sec.
Leave to dry, examine under a microscopy.

2.
3.
4.
5.

6.
7.
8.
9.

Gram-staining

Staining in cuvettes/ automate

Staining time
1 min, 30 sec
30 sec
3 min

4
5

20 sec
5-10 sec

6
7
8
9

30 sec
1 min
1 min
5 min

Effective measures to be taken to protect against infection in line with

laboratory guidelines.

Instructions for disposal

Used solutions and solutions that are past their shelf-life must be disposed of
as special waste in accordance with local guidelines. If you have more
detailed questions regarding disposal, these can be forwarded by e-mail:
retrologistik@merck.de . Within the EU the currently applicable regulations on
the basis of Directive 67/548/EEC of the Council for harmonization of the legal
and administrative regulations apply for the classification, packaging, and
labelling of hazardous substances.

Individual reagents

If the immersion method results in coloration, it is recommended to dilute the

crystal violet solution 1: 3 with demineralized water.

Step
1
2
3

Protection against infection

Solution
crystal violet
running tap water
Lugol's solution
stabilized
running tap water
decolorisation solution
running tap water
safranin solution
running tap water
(dry at 50C)

Cat. No. 1.00983 Ethanol GR

Cat. No. 1.09215 Ziehl-Neelsen's carbol
fuchsin solution

1 l, 2.5 l
100 ml, 500 ml, 2.5 I

Notes

Filter after
three runs
Discard
after 5 runs

Auxiliaries
Cat. No. 1.07961
Cat. No.1.15577
Cat. No.1.04699
Cat. No. 1.06965
Cat. No.1.15525

Entellan new
(rapid covering agent)
Immersion oil
acc. to ISO 8036 mod.
Immersion oil
Oil of cedar wood
RINGER-tablets

100 ml, 500 ml

100 ml
100 ml, 500 ml
100 ml, 500 ml
1 pack (100 tabl.)

Safety classification
Cat. No. 1.11885.0001
Solution 1 or Cat. No. 1.09218, Solution 2 or Cat. No, 1.00567 or Cat. No.
1.09261, Solution 3/4 or Cat. No.1.10218 , Solution 5 or Cat. No. 1.09217
Please observe the hazard classification on the label and the information
given in the safety data sheet.
The Merck safety data sheet is available on the Internet and on request.

Main product components

Cat. No. 1.11885.0001
Solution 1 or Cat. No. 1.09218
C.I. 42555
10g/l
4g/l
C6H5OH
1l = 0,99 kg
Solution 2 or Cat. No, 1.00567
PVP-Iodine
50 g/l
KI
10 g/l
1l =
1.02 kg

or Cat. No. 1.09261

I2
3.4 g/l
Kl
6.8 g/l
1l=
1.01 kg
Solution 3/4 or Cat. No.1.10218

C2H6O
C3H60
1l =

634 g/l
159 g/l
0.79 kg

Solution 5 or Cat. No. 1.09217

C.I. 50240
1,8 g/l
1l =
0,98 kg

Status: April 2009

Merck KGaA, 64271 Darmstadt, Germany

Tel. +49 (0) 61 51 72-2440, www.merck.de
____________________________________________________