In Vitro Diagnostic Medical Device
For professional use only
Methylene blue Gurr®
for microscopical staining (C.I. 52015)
Cat. No
Pack Type
Pack Size
340484B
Glass Bottle
25 g
Composition
C.I. Nr. 52015
C16H18ClN3S · 2-3 H2O
M = 373.90 g/mol (·3H2O)
Dye content
min. 82%
Intended Use(s)
Dye for staining of blood smears to differentiate organisms by
morphological and visualize protozoa’s/ blood parasites and
for bacteriological staining to detect acid-fast microorganisms
Evaluate the result by comparing it to positive control slides
When protozoa’s/ blood parasites and acid-fast bacteria are
found in the material examined, further investigations in a
special laboratory are indicated.
Review of the samples helps in determining the need for
ancillary studies.
An initial review ot the patient´s clinical background is
necessary to use in conjunction with the result of the staining
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 such
as HCl-ethanol. They remain red after counterstaining with
methylene blue, whereas the microorganisms susceptible to
acid take on the blue.
Reagent
Cat. No Description
34048 Methylene blue (C.I. 52015)
Haematology
34197 Eosin Y (C.I. 45380)
20847 Methanol Analar Normapur
Reagent Ph.Eur.
24388 Glycerol Bidistilled 99,5%
Normapur
36311 Weise buffer tablets pH 6,8
1.09468 Weise buffer tablets pH 7,2
Conn´s Biological stains 10th edition, R.W. Horobin,
J.A. Kiernan
Bacteriology
20821 Ethanol Absolute Normapur
Analytical Reag
26668 Potassium Hydroxide pellets
Normapur AR
1.09215 Ziehl-Neelsen carbol fuchsin
solution
1.00327 Hydrochroric acid in ethanol
Characteristics and performances
Preparation
Samples derived from the human body
References:
Manual of Clinical Microbiology 6th edition , Patrick R. Murray
Haematological 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 such
as pH of the solutions and buffer solution, buffer substances,
fixation, staining time.
The haematological staining technique is used for
visualisation of blood parasites/ protozoa’s in blood. The
nuclei of blood parasites/ protozoa’s 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 Ziehl-Neelsen staining, the test
Pack Size
25 g
25 g, 100 g, 1 kg
1 l, 2,5 l, 5 l
250 ml, 500 ml, 1 l,
2,5 l, 5 l, 20 l
1 pack (100 tabs)
1 pack (100 tabs)
1 l, 2,5 l, 5 l
500 g, 1 kg, 5 kg
500 ml, 2,5 l
1 l, 2,5 l
Haematology
1. May-Grünwald staining solution
Mix 0.5 g Eosin Y and 0.5 g methylene blue in 100 ml
distilled water. Filter. 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 60°C in 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.
4. Buffer solution
Dissolve 1 buffer tablet* in 1l distilled water.
*1.09468 or 36311 depending on required reaction colour
VWR International bvba
Researchpark Haasrode 2020
Geldenaaksebaan 464
3001 Leuven
Belgium
http://www.vwr.com
Rev. Date: March
2011
Page 1 of 4
In Vitro Diagnostic Medical Device
For professional use only
Methylene blue Gurr®
for microscopical staining (C.I. 52015)
Bacteriology
1. Löffler's methylene blue solution
Dissolve 0.3 g methylene blue with 30 ml of 95% ethanol
and add this to 100 ml of 0.1% aqueous KOH.
2.Aqueous 0.1% KOH solution
Dissolve 1 g KOH with stirring in 1 l distilled water
Sample material and preparation
For professional use only.
Haematology
Air-dried blood smears derived from the human body
Films are made by placing a drop of the samples on one end
of a slide, and using a spreader slide to disperse the sample
over the slide's length. The aim is to get a region where the
cells are spaced far enough apart to be counted and
differentiated.The slide is left to air dry
In order to avoid errors, the staining process must be carried
out by qualified personnel.
National guidelines for work safety and quality assurance must
be followed.
Microscopes equipped according to the standard must be
used.
Suitable instruments must be used for taking samples and for
their preparation. Follow the manufacturer’s instructions for
application/use.
All samples must be clearly labelled.
Bacterology
Using an ignited loop, transfer a quantity of specimen 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
Heat –fixation:
Dry the smear at room temperature.Grip the slide and pass
through the flame of a Bunsen Burner several times to heatkill and adhere the organism to the slide
It is also possible to fix the smears in an oven at
100-110°C for 20 min.
Leave to cool and stain.
All samples must be clearly labelled.
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.
Procedure
Haematology
Fixation is carried out in the first staining step with undiluted
May-Grünwald solution
Staining rack
1. On to each fresh, dried film, pipette just enough
May-Grünwald 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 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 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 methylene
blue solution or for 1 min with a diluted solution
(dilution 1:10 (1+9) with distilled water)
6. Wash well with tap water.
7. Dry and examine under the microscope.
Allow the specimens to dry and, if necessary mount
Dehydrate histological specimens (ascending alcohol series)
Specimens for use in histology and cytology must be
completely anhydrous prior to being mounted. Xylene should
be added as a final stage in order to prevent turbidity brought
about by solvents containing water.
VWR International bvba
Researchpark Haasrode 2020
Geldenaaksebaan 464
3001 Leuven
Belgium
http://www.vwr.com
Rev. Date: March
2011
Page 2 of 4
In Vitro Diagnostic Medical Device
For professional use only
Methylene blue Gurr®
for microscopical staining (C.I. 52015)
To carry out the mounting process, drop approximately 0.5 ml
mounting agent onto a horizontal slide using a glass rod. This
fills the space between slide and coverglass. As soon as the
specimen has been covered with a homogeneous solution,
cover with a coverglass, taking care to avoid air bubbles.
Allow to harden over a period of 20-30 minutes in a horizontal
position.
Result
The microscope used should meet the requirements of a
medical diagnostic laboratory
Haematology
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.
Diagnostics
Diagnoses are only to be made by authorised and trained
persons. Valid nomenclatures must be used.
Further tests must be selected and implemented according to
recognised methods.
Storage
The dye must be stored at +5°C to +30°C.
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 +5°C to +30°C. The
bottles must be kept tightly closed at all times.
Auxiliary reagents
Cat. No Description
36126 Microil Immersion oil
tropical grade
36104 Microil Immersion Oil
36102 Lenzol Immersion oil Gurr
36194 Fractoil Synthetic
Immersion Oil
36125 DePeX® mounting medium
36029 DPX mountant
Pack Size
100 ml
100 ml, 500 ml
100 ml
500ml
500ml
100ml, 500ml
Precautioniary measures on health hazards
Effective measures must be taken to protect against infection
in line with laboratory guidelines.
Futher tests must be selected and implemented acc. to
recognized methods for identification
Physical Hazard classification
References:
*Manual of Clinical Microbiology 6th edition , Patrick R. Murray
*Conn´s Biological stains 10th edition, R.W. Horobin,
J.A. Kiernan
Instructions for environmental disposal
Please observe the hazard classification on the label and the
information given in the safety data sheet.
The VWR safety data sheet is available on the Internet.
Used solutions and solutions that are past their shelf-life must
be disposed of as special waste according to local disposal
guidelines.VWR International can provide technical support for
local disposal solutions.
VWR International bvba
Researchpark Haasrode 2020
Geldenaaksebaan 464
3001 Leuven
Belgium
http://www.vwr.com
Rev. Date: March
2011
Page 3 of 4
In Vitro Diagnostic Medical Device
For professional use only
Methylene blue Gurr®
for microscopical staining (C.I. 52015)
VWR International bvba
Researchpark Haasrode 2020
Geldenaaksebaan 464
3001 Leuven
Belgium
http://www.vwr.com
Rev. Date: March
2011
Page 4 of 4