In Vitro Diagnostic Medical Device For professional use only Safranine O Gurr® for microscopical staining Cat. No Pack Type Pack Size Using an ignited loop, transfer a quantity of specimen, which 343122N Glass Bottle 25 g may be a body fluid, exudate, pus, or a liquid or solid culture, on to a degreased slide. Then distribute the specimen either Composition directly or after adding 1-2 drops of physiological saline C.I. No. 50240 >80% solution. After drying in air, heat-fix the smear C H ClN Heat –fixation: 20 19 4 Dry the smear at room temperature.Grip the slide and pass M = 350.85 g/mol through the flame of a Bunsen Burner several times to heat- kill and adhere the organism to the slide Intended Use(s) Leave to cool and stain. Dye for Gram stain procedures .Gram staining is a method All samples must be clearly labelled. used for direct microscopical examination to differentiate bacterial species into two large groups (Gram-positive and Gram-negative) based on the physical properties of their cell Preparation walls Safranin O stock solution The Gram stain provides prelimary identification information Dissolve 2.5 g safranin O Gurr® in 100 ml of 96% ethanol. and is not a substitute for culture studies for the diagnosis of For use, 10 ml of solution should be diluted with 90 ml of infectious diseases in human distilled water. Futher tests must be selected and implemented acc. to recognized methods for identification Staining on a staining bench Samples are derived from the human body, and heat fixed on 1. Completely cover the slide with solution 1 : crystal violet a degreassed slide and examinated under microscope solution. Stain for 1 min, pour off. 2. Carefully rinse with solution 2: Lugol's stabilized solution. Reference: 3. Completely cover the slide with solution 2: Lugol's *Manual of Clinical Microbiology 6th edition , Patrick R. Murray stabilized solution. Allow to act for 1 min. Characteristics and performances 4. Carefully rinse with distilled water for about 5 sec. Differentiation of gram-positive and gram-negative bacteria. 5. Swirl the slide for about 10-15 sec in solution 3 or 4; In the staining procedure, aniline dyes in the cell walls of decolourisation solution. Stop swirling when no more dye bacteria are bound under the influence of iodine to iodine-dye is released complexes. and the smear appears greyish-blue. In the case of gram-positive bacteria, such complexes cannot 6. Carefully rinse with distilled water for about 5 sec. be released from the cell with decolourizing agents like 7. Completely cover the slide with solution 5: safranin alcohol or acetone; the cell retains its blue-violet stain. solution. Stain for 1 min. Gram-negative bacteria on the other hand release the iodine- 8. Carefully rinse with distilled water for about 5 sec. dye complex and the cell can be counter-stained pink to red. 9. Leave to dry, examine under a microscope. Staining in cuvettes/ automate Reagent Step Staining time Solution Notes Cat. No Description Pack Size 1 1 min, 30 sec crystal violet 34312 Safranin O (C.I. 50240) Gurr® 25 g 2 30 sec running tap water 10476 Ethanol 96%, extra pure 1 l, 2,5 l 3 3 min Lugol's stabilized Filter after 35190 Lugol's Iodine Solution 250ml solution three runs 1.10218 Gram's Decolorisation Solution 500ml, 2,5l 4 20 sec running tap water 1.09218 Gram's Crystal Violet Solution 500ml, 2,5l 5 5 – 10 sec decolourisation Discard after solution 5 runs Sample preparation 6 30 sec running tap water 7 1 min safranin solution For professional use only VWR International bvba Researchpark Haasrode 2020 Geldenaaksebaan 464 3001 Leuven Belgium http://www.vwr.com Rev. Date: March 2011 Page 1 of 3 In Vitro Diagnostic Medical Device For professional use only Safranine O Gurr® for microscopical staining 8 1 min running tap water National guidelines for work safety and quality assurance 9 5 min (dry at 50°C) must be followed. Microscopes equipped according to the standard must be Result used. The microscope used should meet the requirements of a All samples must be clearly labelled. medical diagnostic laboratory Suitable instruments must be used for taking samples and Gram-positive microorganisms blue-violet their preparation; manufacturer instructions for application/use Gram-negative microorganisms pink to red must be followed. Gram-positive microorganisms stained blue-violet Diagnostics Staphylococcus, Streptococcus, Enterococcus, (cocci) and Diagnoses are only to be made by authorised and trained Bacillus, Corynebacterium, Nocardia, Clostridium, persons. Valid nomenclatures must be used. Actinobacteria, and Listeria (rods) Further tests must be selected and implemented according to recognised methods. Storage The dye must be stored at +5°C to +30°C. 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. Staphylococcus aureus Shelf life Gram-negative microorganisms stained pink to red Escherichia coli, Salmonella, Shigella, and other The bottles must be kept tightly closed at Enterobacteriaceae, Pseudomonas, (bacilli), Neisseria,(cocci) all times. The reagents must be used by the expiry date stated Auxiliary reagents Cat. No Description Pack Size 36126 Microil Immersion Oil 100 ml tropical grade 36104 Microil Immersion Oil 100 ml, 500 ml 36102 Lenzol Immersion Oil Gurr 100 ml Pseudomonas aeruginosa 36194 Fractoil Synthetic 500 ml Immersion Oil The Gram stain provides prelimary identificaion information and is not a substitute for culture studies Futher tests must be selected and implemented acc. to Precautioniary measures on health hazards recognized methods for identification Effective measures must be taken to protect against infection in line with laboratory guidelines. Reference: *Manual of Clinical Microbiology 6th edition , Patrick R. Murray Physical Hazard classification Please observe the hazard classification on the label and the Instructions for use information given in the safety data sheet. For professional use only. The VWR safety data sheet is available on the Internet. In order to avoid errors, the staining process must be carried out by an expert. VWR International bvba Researchpark Haasrode 2020 Geldenaaksebaan 464 3001 Leuven Belgium http://www.vwr.com Rev. Date: March 2011 Page 2 of 3 In Vitro Diagnostic Medical Device For professional use only Safranine O Gurr® for microscopical staining Instructions for environmental disposal 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 3 .