[+1] 800.221.6058 United States Directions For Use for the following: [+1] 360.260.2779 International Set, Gram Stain Set with Carbolfuchsin, Gram , Gram Stabilized Iodine, Gram Decolorizer, Gram Safranin, Gram Carbolfuchsin

INTENDED USE SPECIMEN PROCESSING Alpha-Tec Systems, Inc. Gram Stain Set, Gram Stain Set with 1. Make a thin smear of the specimen over an area of 2-3 sq cm on a Carbolfuchsin, stains and reagents are recommended for use in glass slide. Heat fix by warming on a slide warmer (65-75ºC) for no qualitative procedures to assist in identification and classification of more than 2 hours. If a Bunsen burner is used, pass the slide slowly bacterial organisms. 3 times over its cone of heat. Do not scorch. Alternatively, methanol can be used to fix the slide. Flood the slide with methanol for 1-2 SUMMARY minutes and air-dry. In 1884, Hans Christian Joachim Gram improved a method with 2. Flood the smear with Gram Crystal Violet Stain for 1 minute. the intention to differentiate pneumococci from lung tissue. In his 3. Rinse with deionized or distilled water and drain. procedure, he noticed that certain retained the stain while 4. Flood the smear with Gram Stabilized Iodine for 1 minute. others did not. However, he did not realize how this method correlated 5. Rinse with deionized or distilled water and drain. to the unique attributes of the cells nor its importance as a preliminary 6. Decolorize with Gram Decolorizer until solvent running from the means to classify bacteria taxonomically and phenotypically. The Gram slide is colorless (3-60 seconds). stain has since become one of the relied upon methods employed in 7. Rinse with deionized or distilled water and drain. laboratories for differentiating bacteria. 8. Flood the smear with Gram Safranin or Gram Carbolfuchsin for 1 minute. Upon passing through the cell wall of the organism, Crystal Violet and 9. Rinse with deionized or distilled water and allow to air dry. Iodine will form a complex. Some types of bacteria will retain this 10. Examine under low power (10-40x), then under oil immersion complex after a wash with an organic solvent, such as those found in (100x) for presence of Gram-positive and Gram-negative the Gram Decolorizer, while others will not. Those that retain the Crystal organisms. Violet are known as Gram-positive (purple in color) while those that only retain the , Gram Safranin or Gram Carbolfuchsin, are EXPECTED RESULTS known as Gram-negative (pink in color). Positive Result: Gram-positive organisms stain purple. Negative Result: Gram-negative organisms stain pink. It has been generally accepted that the higher (e.g. peptidoglycan) content of Gram-negative cell walls is the cause of the differential LIMITATIONS OF PROCEDURES staining. Additionally, due to the unique properties of particular bacteria, 1. Avoid overexposure of the decolorizer to the slide as it may remove some species will have cell walls that easily burst under these staining color from both Gram-negative and Gram-positive bacteria. conditions while others will have irregular lipid content in cell walls 2. Avoid excessive washing as it may cause a heightened depending on the age of the culture. Due to these variables, this method decolorization effect. may give ambiguous results and additional testing should be conducted 3. Smears should not be too thick. After air-drying, examine under a to confirm identification. microscope. Spread the smear so as to prevent overcrowding as this may inhibit proper decolorization. FOR IN VITRO DIAGNOSTIC USE ONLY 4. The Gram stain offers a preliminary form of identification. However, all results should be confirmed with cultures and/or additional PRECAUTIONS methodologies. Gram Crystal Violet Stain, Gram Stabilized Iodine, Gram Decolorizer, 5. For patient slides, proper heat fixation must be used to prevent Gram Safranin, and Gram Carbolfuchsin may be harmful if swallowed changes to the cell wall and chemical structure of the cell wall. Care and may cause irritation to skin, eyes and respiratory tract. Gram Crystal must be taken to prevent heating the slide too long or too hot. A Violet Stain, Gram Decolorizer and Gram Safranin are flammable. Keep microorganism that is disrupted by excessive heat will not react to away from heat and flame. Avoid breathing vapor and eye/skin contact. Gram staining as expected. 6. Caution should be used in interpretation as results may be STABILITY AND STORAGE inconclusive due to: morphology of the organism (e.g. acid-fast This product is ready to use, and no further preparation is necessary. mycobacteria), age of the culture, interference from autolytic Store in original container at 15-30ºC. Store away from heat and flame enzymes (e.g. S. pneumonia), and cultures that have been treated sources. with antibiotics, either in the host or from media. 7. The sensitivity of the direct smear is lower than that of culture USER QUALITY CONTROL methods. Therefore, cultures should be performed on all Any reagent showing a change of color or other sign of deterioration specimens. should be discarded. Quality controlled organisms should be utilized to verify procedures and reagents as appropriate for your laboratory’s SPECIFIC PERFORMANCE CHARACTERISTICS applicable regulatory agency or local procedural guidelines. These products have been tested using QC1™ Gram Slides and have been found to yield acceptable stain results. Appropriate testing should SPECIMEN COLLECTION AND PREPARATION be performed in accordance with established laboratory quality control Specimens should be collected and handled following recommended procedures. If aberrant quality control results are noted, patient results guidelines. should not be reported.

PROCEDURE BIBLIOGRAPHY Materials provided: The Gram Stain Set contains Gram Crystal Violet 1. Gram. 1884. Fortschritte der Medicin. 2:185-189. Stain, Gram Stabilized Iodine, Gram Decolorizer, Gram Safranin. Gram 2. Davies, J. A., et al. 1983. J. Bacteriol. 156(2):837-845. Stain Set with Carbolfuchsin replaces Gram Safranin with Gram 3. Kinyoun, J.J. 1915. Am. J. Public Health. 5:867-870. Carbolfuchsin. Each component is also available individually. 4. Mangels, et al. 1984. Microbiol. Infect. Dis. 2:129-137. Materials not provided: Pipettes, glass slides, microscope, immersion 5. Magee, C.M., G. Rodenheaver, M.T. Edgerton, and R.F. Edlich. oil, deionized or distilled water, Bunsen burner or slide warmer, QC1™ 1975. Am. J. Surg. 130:341-346. Gram Slide (#0003230).

L003576.C.2 // CC20-132, Effective Date: 08 Oct 2020 // ©2020 Alpha-Tec Systems, Inc. All rights reserved. 1 [+1] 800.221.6058 United States Directions For Use for the following: [+1] 360.260.2779 International Gram Stain Set, Gram Stain Set with Carbolfuchsin, Gram Crystal Violet, Gram Stabilized Iodine, Gram Decolorizer, Gram Safranin, Gram Carbolfuchsin

CONTACT Alpha-Tec Systems, Inc. offers a complete line of reagents, stains, and QC1™ Quality Control Slides for AFB, Parasitology, Bacteriology, and Mycology processing, as well as O&P collection systems and concentration devices for Parasitology. For Technical Assistance, email [email protected], and for Customer Service, email [email protected], or call either [+1] 800.221.6058 (USA) or [+1] 360.260.2779 between 8AM and 4PM Monday through Friday, Pacific Time.

WARRANTY This product is warranted by Alpha-Tec Systems, Inc. to perform as described in the labeling and literature supplied. Alpha-Tec Systems, Inc. disclaims any implied warranty or merchantability or fitness for any other purpose, and in no event shall Alpha-Tec Systems, Inc. be liable for any consequential damages arising out of aforesaid express warranty.

TRADEMARKS QC1™ is a trademark of Alpha-Tec Systems, Inc., 1311 SE Cardinal Court, Suite 170, Vancouver, WA 98683 USA.

PRODUCT CODES 0003576 Gram Stain Set, 4 x 250 ml 0003579 Gram Stain Set with Carbolfuchsin, 4 x 250 ml 0004702 Gram Crystal Violet Stain, 250 ml 0004702C Gram Crystal Violet Stain, 8 x 250 ml 0004703 Gram Stabilized Iodine, 250 ml 0004704 Gram Decolorizer, 250 ml 0004704C Gram Decolorizer, 8 x 250 ml 0004705 Gram Safranin, 250 ml 0004705C Gram Safranin, 8 x 250 ml 0004709 Gram Carbolfuchsin Counterstain

L003576.C.2 // CC20-132, Effective Date: 08 Oct 2020 // ©2020 Alpha-Tec Systems, Inc. All rights reserved. 2