In Vitro Diagnostic Medical Device For professional use only
Methylene blue Gurr® for microscopical staining (C.I. 52015)
Cat. No Pack Type Pack Size material has to be heated with carbol fuchsin solution to 340484B Glass Bottle 25 g produce the mycolic acid fuchsin compound. Once stained, acid fast mycobacteria keep their colouring Composition even after treatment with strong decolourizing solutions such C.I. Nr. 52015 as HCl-ethanol. They remain red after counterstaining with C H ClN S · 2-3 H O methylene blue, whereas the microorganisms susceptible to 16 18 3 2 acid take on the blue. M = 373.90 g/mol (·3H2O) Dye content min. 82% Reagent Intended Use(s) Cat. No Description Pack Size Dye for staining of blood smears to differentiate organisms by 34048 Methylene blue (C.I. 52015) 25 g morphological and visualize protozoa’s/ blood parasites and for bacteriological staining to detect acid-fast microorganisms Haematology Evaluate the result by comparing it to positive control slides 34197 Eosin Y (C.I. 45380) 25 g, 100 g, 1 kg When protozoa’s/ blood parasites and acid-fast bacteria are 20847 Methanol Analar Normapur 1 l, 2,5 l, 5 l found in the material examined, further investigations in a Reagent Ph.Eur. special laboratory are indicated. 24388 Glycerol Bidistilled 99,5% 250 ml, 500 ml, 1 l, Review of the samples helps in determining the need for Normapur 2,5 l, 5 l, 20 l ancillary studies. 36311 Weise buffer tablets pH 6,8 1 pack (100 tabs) An initial review ot the patient´s clinical background is 1.09468 Weise buffer tablets pH 7,2 1 pack (100 tabs) necessary to use in conjunction with the result of the staining Bacteriology Samples derived from the human body 20821 Ethanol Absolute Normapur 1 l, 2,5 l, 5 l Analytical Reag References: 26668 Potassium Hydroxide pellets 500 g, 1 kg, 5 kg Manual of Clinical Microbiology 6th edition , Patrick R. Murray Normapur AR 1.09215 Ziehl-Neelsen carbol fuchsin Conn´s Biological stains 10th edition, R.W. Horobin, solution 500 ml, 2,5 l J.A. Kiernan 1.00327 Hydrochroric acid in ethanol 1 l, 2,5 l
Characteristics and performances Preparation Haematological methods Haematology The typical colour of cell nuclei, namely purple, is due to 1. May-Grünwald staining solution molecular interaction between eosin y and the methylene Mix 0.5 g Eosin Y and 0.5 g methylene blue in 100 ml blue/azure B-DNA complex. distilled water. Filter. Dry filtrate. Wash residue and dry. The staining result can be influenced by several factors such Dissolve in 50 ml methanol, this is the stock solution. as pH of the solutions and buffer solution, buffer substances, 2. Giemsa's azur eosin methylene blue solution fixation, staining time. Dissolve 0.76 g Giemsa's azur eosin methylene blue in The haematological staining technique is used for 50 ml glycerol and heat for 3 h at 60°C in a water bath, visualisation of blood parasites/ protozoa’s in blood. The add 50 ml methanol, leave to stand for 5 days and filter. nuclei of blood parasites/ protozoa’s appear red under the 3. Diluted Giemsa's solution microscope. Dilute 10 ml Giemsa's azur eosin-methylene blue solution with 190 ml buffer solution, mix well, allow to stand for 10 Bacteriological methods min and filter, if necessary. Mycobacteria are difficult to stain because of the high 4. Buffer solution proportion of lipid and wax in their cell walls. Up to now, in Dissolve 1 buffer tablet* in 1l distilled water. order to carry out the classical Ziehl-Neelsen staining, the test *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)
In order to avoid errors, the staining process must be carried out by an expert. Bacteriology National guidelines for work safety and quality assurance 1. Löffler's methylene blue solution must be followed. Dissolve 0.3 g methylene blue with 30 ml of 95% ethanol Microscopes equipped according to the standard must be and add this to 100 ml of 0.1% aqueous KOH. used. 2.Aqueous 0.1% KOH solution Dissolve 1 g KOH with stirring in 1 l distilled water Procedure Haematology Sample material and preparation Fixation is carried out in the first staining step with undiluted For professional use only. May-Grünwald solution Staining rack Haematology 1. On to each fresh, dried film, pipette just enough Air-dried blood smears derived from the human body May-Grünwald solution to cover the blood film Films are made by placing a drop of the samples on one end (usually 10 drops or more) and let react for 3 min. of a slide, and using a spreader slide to disperse the sample 2. Add an equal amount of distilled water, mix and over the slide's length. The aim is to get a region where the stain for 1 min. cells are spaced far enough apart to be counted and 3. Pour off fluid and without washing add about 10 differentiated.The slide is left to air dry drops of diluted buffered Giemsa solution, stain for 5 In order to avoid errors, the staining process must be carried - 60 min (try first for 10 -15 min). out by qualified personnel. 4. Rinse with buffer solution. National guidelines for work safety and quality assurance must 5. Dry and examine under the microscope. be followed. Microscopes equipped according to the standard must be Bacteriology used. Staining rack Suitable instruments must be used for taking samples and for 1. Flood specimens completely with carbol-fuchsin their preparation. Follow the manufacturer’s instructions for solution. Carefully heat 3 times from below with a application/use. bunsen burner to steaming and keep hot for 5 min. Do not allow the stain to boil. All samples must be clearly labelled. 2. Wash with tap water until no further colour is given off. Bacterology 3. Cover completely with decolourizing solution and, Using an ignited loop, transfer a quantity of specimen on to a depending on the thickness of the specimen, allow degreased slide. Then distribute the specimen either directly to stand for 15 – 30 sec. or after adding 1-2 drops of physiological saline solution. After 4. Wash immediately with tap water. drying in air, heat-fix the smear 5. Counterstain by flooding for 30 sec in methylene Heat –fixation: blue solution or for 1 min with a diluted solution Dry the smear at room temperature.Grip the slide and pass (dilution 1:10 (1+9) with distilled water) through the flame of a Bunsen Burner several times to heat- 6. Wash well with tap water. kill and adhere the organism to the slide 7. Dry and examine under the microscope. It is also possible to fix the smears in an oven at 100-110°C for 20 min. Allow the specimens to dry and, if necessary mount Leave to cool and stain. Dehydrate histological specimens (ascending alcohol series) All samples must be clearly labelled. 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, Diagnostics cover with a coverglass, taking care to avoid air bubbles. Diagnoses are only to be made by authorised and trained Allow to harden over a period of 20-30 minutes in a horizontal persons. Valid nomenclatures must be used. position. Further tests must be selected and implemented according to recognised methods.
Result Storage The microscope used should meet the requirements of a medical diagnostic laboratory The dye must be stored at +5°C to +30°C. The dye must be used by the expiry date Haematology stated. Nuclei red to violet Lymphocytes plasma blue, azur granules purple to red Monocytes plasma dove-blue Shelf life Neutrophilic granulocytes granules light violet After the first opening of the bottle the Eosinophilic granulocytes granules red to grey-blue contents can be used up to the expiry Basophilic granulocytes granules dark violet date when stored at +5°C to +30°C. The Thrombocytes violet bottles must be kept tightly closed at all times. Erythrocytes red Blood parasites nuclei bright red Auxiliary reagents Cat. No Description Pack Size Bacteriology 36126 Microil Immersion oil 100 ml Mycobacteria red tropical grade Background light blue 36104 Microil Immersion Oil 100 ml, 500 ml 36102 Lenzol Immersion oil Gurr 100 ml A positive finding is reported as "acid fast bacteria detected" 36194 Fractoil Synthetic 500ml and a negative finding is reported as "acid fast bacteria not Immersion Oil detected". It is not possible to state whether there are 36125 DePeX® mounting medium 500ml tuberculosis bacteria or other "atypical" bacteria. 36029 DPX mountant 100ml, 500ml It is also impossible to state whether these mycobacteria are still capable of reproduction or are already dead. Precautioniary measures on health hazards When acid-fast bacteria are found in the material examined, Effective measures must be taken to protect against infection further investigations in a special laboratory are indicated. in line with laboratory guidelines.
Futher tests must be selected and implemented acc. to recognized methods for identification Physical Hazard classification Please observe the hazard classification on the label and the References: information given in the safety data sheet. *Manual of Clinical Microbiology 6th edition , Patrick R. Murray The VWR safety data sheet is available on the Internet. *Conn´s Biological stains 10th edition, R.W. Horobin, Instructions for environmental disposal J.A. Kiernan 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