1.15943.

0025
1.15943.0100
1.15943.1000
1.15943.5000

4. Buffer solution
Dissolve 1 buffer tablet* in 1l distilled water.
* 111374 or 109468 depending on required reaction colour

Microscopy
Methylene blue (C.I. 52015) Certistain
Dye for staining of blood smears containing protozoans/
blood parasits and for bacteriological staining

Bacteriology
1. Fixation
Fixation is carried out over the flame of a Bunsen burner (2-3 times,
avoiding excessive heating).
It is also possible to fix the smears in an oven at 100-110 C for 20 min.
2. Lffler's methylene blue solution
Dissolve 0.3 g methylene blue Certistain with 30 ml of 95% ethanol and
add this to 100 ml of 0.1% aqueous KOH.
3. Aqueous 0.1% KOH solution
Dissolve 1 g KOH under stiring in 1 l aqua dest..

Staining procedure

Principle
Hematological methods
The typical colour of cell nuclei, namely purple, is due to molecular interaction
between eosin y and the methylene blue/azure B-DNA complex.
The staining result can be influenced by several factors as pH of the solutions
and buffer solution, buffer substances, fixation, staining time.
The hematological staining technique is used for visualisation of blood parasits/
protozoans in blood. The nuclei of blood parasits/ protozoans appear red under
the microscope.
Bacteriological methods
Mycobacteria are difficult to stain because of the high proportion of lipid and
wax in their cell walls. Up to now, in order to carry out the classical ZiehlNeelsen staining, the test material has to be heated with carbol fuchsin solution
to produce the mycolic acid fuchsin compound.
Once stained, acid fast mycobacteria keep their colouring even after treatment
with strong decolourizing solutions as HCl-ethanol. They remain red after
counterstaining with methylene blue, whereas the microorganisms susceptible
to acid take on the blue.

Sample material
Hematology
Air-dried blood smears
Bacterology
Heat-fixed smears of sputum, FNAB, lavages, imprints, body fluids, exudates,
pus, liquid and solid cultures, histological sections.

Reagents
Cat.No. .15943 Methylene blue (C.I. 52015) Certistain
25 g, 100 g
Colour Index No.
C.I. 52015
Colour Index Name:
Basic Blue 9, Solvent blue 8
Hematology
Cat.No. 115935 Eosin Y (C.I. 45380) Certistain
Cat.No. 109203 Giemsa's azur eosin
methylene blue
Cat.No. 106009 Methanol for analysis
EMSURE ACS,ISO,Reag. Ph Eur
Cat.No. 104092 Glycerol
Cat.No. 109468 Weise buffer tablets pH 7.2
Cat.No. 111374 Weise buffer tablets pH 6.8

25 g, 100 g
25 g, 100 g
1 l, 2.5 l
1 l, 2.5 l
1 pack (100 tabs)
1 pack (100 tabs)

Bacteriology
Cat.No. 109215 Ziehl-Neelsen carbol fuchsin solution
Cat.No. 100327 Hydrochloric acid in ethanol
Cat.No. 100983 Ethanol for analysis
EMSURE ACS, ISO, Reag. Ph Eur
Cat.No. 105033 Potassium hydroxide

500 ml, 2.5 l

1 l, 2.5 l
1 l, 2.5 l
500 g

Preparation
Hematology
1. May-Grnwald staining solution
Mix 0.5 g Eosin Y Certistain and 0,5 g methylene blue Certistain in 100 ml
distilled water. Filtrate. Dry filtrate. Wash residue and dry. Dissolve in 50 ml
methanol, this is the stock solution.
2. Giemsa's azur eosin methylene blue solution
Dissolve 0.76 g Giemsa's azur eosin methylene blue in 50 ml glycerol and heat
for 3 h at 60C on a water bath, add 50 ml methanol, leave to stand for 5 days
and filter.
3. Diluted Giemsa's solution
Dilute 10 ml Giemsa's azur eosin-methylene blue solution with 190 ml buffer
solution, mix well, allow to stand for 10 min and filter, if necessary.

Hematology
Fixation is carried out in the first staining step with undiluted May-Grnwald
solution
Staining rack
1.
Onto each fresh, dried film, pipette just enough May-Grnwald
solution to cover the blood film (usually 10 drops or more) and let
react for 3 min.
2.
Add an equal amount of distilled water, mix and stain for 1 min.
3.
Pour off fluid and without washing add about 10 drops of diluted
buffered Giemsa solution, stain for 5 - 60 min (try first for 10 -15 min).
4.
Rinse with buffer solution.
5.
Dry and examine under the microscope.
Bacteriology
Staining rack
1.
Flood specimens completely with Tb-color modified carbol-fuchsin
solution. Carefully heat 3 times from below with a bunsen burner to
steaming and keep hot for 5 min. Do not allow the stain to boil.
2.
Wash with tap water until no further colour is given off.
3.
Cover completely with Tb-color modified decolourizing solution and,
depending on the thickness of the specimen, allow to stand for 15
30 sec.
4.
Wash immediately with tap water.
5.
Counterstain by flooding for 30 sec in Tb-color modified methylene
blue solution or for 1 min with a diluted solution (dilution 1:10 (1+9)
with dest. water)
6.
Wash well with tap water.
7.
Dry
Allow the specimens to dry and, if necessary, mount with Entellan new or
Neo-Mount. Dehydrate histological specimens (ascending alcohol series)
and mount with Entellan new or Neo-Mount.

Result
Hematology
Nuclei
Lymphocytes
Monocytes
Neutrophilic granulocytes
Eosinophilic granulocytes
Basophilic granulocytes
Thrombocytes
Erythrocytes
Blood parasites

red to violet
plasma blue,
Azur granules purple to red
plasma dove-blue
granules light violet
granules red to grey-blue
granules dark violet
violet
red
nuclei bright red

Bacteriology
Mycobacteria
Background

red
light blue

A positive finding is reported as "acid fast bacteria detected" and a negative

finding is reported as "acid fast bacteria not detected". It is not possible to
state whether there are tuberculosis bacteria or other "atypical" bacteria.
It is also impossible to state whether these mycobacteria are still capable of
reproduction or are already dead.
When acid-fast bacteria are found in the material examined, further
investigations in a special laboratory are indicated.

Technical note
The microscope used should meet the requirements of a medical diagnostic
laboratory.
Use a full-flow filter to filtrate the diluted staining solution prior to use.

Sample preparations
All samples should be treated using state-of-the-art technology.
All samples must be clearly labelled.
Suitable instruments must be used for taking samples and their preparation;
manufacturer instructions for application/use must be followed.

Diagnostics
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 dye must be stored at +5C to +30C.
The dye must be used by the expiry date stated.

Shelf-life
After the first opening of the bottle the contents can be used up to the expiry
date when stored at +5C to +30C.
The bottles must be kept tightly closed at all times.

Instructions for use

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

Protection against infection

Effective measures to be taken to protect against infection in line with
laboratory guidelines.

Instructions for disposal

The package must be disposed of in accordance with the current disposal
guidelines.
Used solutions and solutions that are past their shelf-life must be disposed of
as special waste in accordance with local guidelines. Information on disposal
can be obtained under the Quick Link Hints for Disposal of Microscopy
Products at www.microscopy-products.com. Within the EU the currently
applicable REGULATION (EC) No 1272/2008 on classification, labelling and
packaging of substances and mixtures, amending and repealing. Directives
67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006
applies.

Auxiliary reagents
Cat. No. 104699
Cat. No. 115577
Cat. No. 107961

Immersion oil
100 ml, 500 ml
Immersion oil
100 ml
acc. to ISO 8036 modified
Entellan new
100 ml, 500 ml

Safety classification
Cat. No. 115943
Please observe the hazard classification on the label and the information given
in the safety data sheet.
The Merck safety data sheet is available at Internet and on request

Main product components

Cat. No. 115943
C.I. 52015
C16H18ClN3S 2-3 H2O
M = 319.86 g/mol (3H2O)
Dye content
min. 82%

Status: June 2014