Iron Hematoxylin Stain Iron Hemotoxylin Mordant

[+1] 800.221.6058 United States Directions For Use for the following: [+1] 360.260.2779 International Iron Hematoxylin Stain Iron Hemotoxylin Mordant

INTENDED USE If conducting the Modified Iron Hematoxylin with Kinyoun Staining The Iron Hematoxylin Stain and Mordant are used in a procedure for Procedure, prepare a 2% phosphotungstic solution and an alcohol permanent staining of intestinal protozoa in fecal specimens preserved ammonium solution as follows: in PROTO-FIX®, PVA (Zn) or SAF. 1. Dilute 2 grams phosphotungstic acid powder in 100 ml distilled SUMMARY water. Solution can be used for one week. Fixing fecal specimens before they are sent to the laboratory has 2. Add 2 ml of 30% ammonia solution to 250 ml of 70% ethanol. become standard good laboratory practice. It is recommended that a Solution can be used for one week. concentration and permanent stain be prepared from a fecal sample. Iron hematoxylin staining produces a permanent stain which will stain Iron Hematoxylin Staining Procedure without Kinyoun Modification intracellular and nuclear structures darker than the cytoplasm, leading to easier identification of parasites based on morphological features. Reagent Timing FOR IN VITRO DIAGNOSTIC USE 70% Ethanol (#0003359) 5 minutes.

Running tap water 2 minutes. Use a constant STABILITY AND STORAGE stream of water into the Iron Hematoxylin Stain and Mordant are stable individually for 12 months container. Remove and drain off when stored at the required temperature. Once working solution is excess liquid by touching edge prepared by combining the reagents as described below, the solution is of slide to absorbent material. stable for one week. Stain should be filtered periodically (after 3-4 runs) Iron hematoxylin working 5 minutes. through coarse filter paper to remove any accumulated debris. Store at solution * (#0003561 & room temperature 15-30ºC. Do not heat. Keep containers well-sealed to #0003562) prevent evaporation. Running tap water 10 minutes. Use a constant stream of water into the USER QUALITY CONTROL A positive parasite smear should be processed with the staining series container. Remove and drain off with each batch of slides or per the regulatory agency used by your excess liquid by touching edge laboratory to verify the quality of the staining reagents and the technique of slide to absorbent material. of the procedure. NOTE: Contact Alpha-Tec Systems, Inc. Sales 70% Ethanol (#0003359) 5 minutes. Remove and drain off department for a complete listing of Quality Control Slides. excess liquid by touching edge of slide to absorbent material. SPECIMEN COLLECTION & PREPARATION 100% Ethanol **(#0003303) 5 minutes. Remove and drain off Refer to the Directions For Use supplied with the O&P Collection Sets, excess liquid by touching edge or available upon request. of slide to absorbent material. 100% Ethanol 5 minutes. Remove and drain off PROCEDURE excess liquid by touching edge Materials Provided: Hematoxylin stain solution and acidic iron solution of slide to absorbent material. mordant. Xylene (#0003342) or 5 minutes. Remove and drain off Materials Not Provided: Staining Reagents and Ethanols, Capillary PRO- CLEAR™ (#0003336) or excess liquid by touching edge Pipettes, Microscope Slides and Coverslips, Absorbent Material ESC-1™ (#0004401) of slide to absorbent material and coverslip with mounting PREPARING COATED SLIDES fluid. Read under oil immersion. NOTE: SPECIMENS PRESERVED IN PROTO-FIX or SAF REQUIRE THE FOLLOWING SLIDE PREPARATION: NOTE: The Xylene substitute, AmeriClear®, does not yield appropriate The CELL-BOND® Slides (#0003257) are ready-to-use coated slides. stain results and should not be used. Unlike Slide Coating Solution, Mayer’s Albumin or PVA, CELL-BOND Slides provide no competitive staining background. This results in a *NOTE: Staining time can vary depending on the intensity desired for clearer microscopic view of the smear. the final stain result. Prepare once weekly.

SPECIMEN PROCESSING **NOTE: 100% Ethanol based reagent alcohol. 1. Gently and evenly spread a portion of the sample over the coated portion of the slide. Use an applicator stick and “chop” the EXPECTED RESULTS specimen, spreading the specimen out to create thick and thin Using the iron hematoxylin staining procedure with organisms fixed in areas. Lay flat with the film up. PROTO-FIX, PVA (Zn), or SAF, the cytoplasm of trophozoites and cysts 2. Allow the slide to remain flat for 1 to 2 minutes. If there is still will be tinged with violet. The nuclear chromatin, chromatoid bodies, excessive liquid on the slide, stand the slides in a slide drying rack erythrocytes and bacteria stain dark purple to black. Helminth eggs and to allow excess liquid to drain. When the specimen on the slide larvae are difficult to distinguish with this staining procedure. stops draining, wipe away the excess liquid on the end. (NOTE: Microsporidia spores are not easily seen with the iron hematoxylin stain. Slides should dry for about 10 minutes. Fecal films dry at different To identify Cryptosporidium parvum, Cyclospora cayetanensis, and rates. If some films are still wet after 10 minutes, they may be air- Isospora belli, follow the Modified Iron Hematoxylin with Kinyoun dried with a fan or a blower. Fecal films can be slightly wet when Staining Procedure. staining begins.)

SOLUTION PREPARATION Prior to beginning the staining procedure (3-4 hour minimum), prepare the working solution by combining equal volumes (1:1) of the Iron Hematoxylin Stain (#0003561) and Iron Hematoxylin Mordant (#0003562). Once prepared, solution can be used for up to one week.

L003561.E // CC19-021, Effective Date: 23 May 2019 // ©2019 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 Iron Hematoxylin Stain Iron Hemotoxylin Mordant

Modified Iron Hematoxylin with Kinyoun Staining Procedure Modified Iron Hematoxylin with Kinyoun Staining Procedure (Canada)

Reagent Timing Reagent Timing 70% Ethanol 5 minutes. Running tap water 2 minutes. Use a constant stream of 70% Ethanol 5 minutes. water into the container. Remove Running tap water 2 minutes. Use a constant stream of and drain off excess liquid by water into the container. Remove touching edge of slide to absorbent and drain off excess liquid by material. touching edge of slide to absorbent TB Kinyoun Carbolfuchsin 5 minutes. material. Stain (#0003524) TB Kinyoun Carbolfuchsin 5 minutes. Running tap water 2 minutes. Use a constant stream of Stain water into the container. Remove Running tap water 2 minutes. Use a constant stream of and drain off excess liquid by water into the container. Remove touching edge of slide to absorbent and drain off excess liquid by material. touching edge of slide to absorbent TB Decolorizer (#0003525) 5 minutes. material. Running tap water 2 minutes. Use a constant stream of TB Decolorizer 6 minutes. water into the container. Remove Running tap water 2 minutes. Use a constant stream of and drain off excess liquid by water into the container. Remove touching edge of slide to absorbent and drain off excess liquid by material. touching edge of slide to absorbent Iron hematoxylin working 5 minutes. material. solution * Iron Hematoxylin Mordant 5 minutes. Running tap water 1 minute. Use a constant stream of water into the container. Remove Running tap water 1minute. Use a constant stream of and drain off excess liquid by water into the container. Remove touching edge of slide to absorbent and drain off excess liquid by material. touching edge of slide to absorbent Phosophotungstic acid 5 seconds. Remove and drain off material. destaining solution excess liquid by touching edge of Iron Hematoxylin Stain* 2 minutes. slide to absorbent material. (#0003562) Running tap water 10 minutes. Use a constant stream Phosophotungstic acid 4 dips. Remove and drain off of water into the container. Remove destaining solution excess liquid by touching edge of and drain off excess liquid by slide to absorbent material. touching edge of slide to absorbent Running tap water 10 minutes. Use a constant stream material. of water into the container. Remove Alcohol ammonium solution 3 minutes. Remove and drain off and drain off excess liquid by excess liquid by touching edge of touching edge of slide to absorbent slide to absorbent material. material. 100% Ethanol ** 5 minutes. Remove and drain off Alcohol ammonium solution 3 minutes. Remove and drain off excess liquid by touching edge of excess liquid by touching edge of slide to absorbent material. slide to absorbent material. 100% Ethanol ** 10 minutes. Remove and drain off 100% Ethanol ** 5 minutes. Remove and drain off excess liquid by touching edge of excess liquid by touching edge of slide to absorbent material. slide to absorbent material. Xylene or PRO-CLEAR or 5 minutes. Remove and drain off 100% Ethanol ** 10 minutes. Remove and drain off ESC-1 excess liquid by touching edge of excess liquid by touching edge of slide to absorbent material and slide to absorbent material. coverslip with mounting fluid. Read Xylene or PRO- CLEAR or 5 minutes. Remove and drain off under oil immersion. ESC-1 excess liquid by touching edge of slide to absorbent material and NOTE: The Xylene substitute, AmeriClear®, does not yield appropriate coverslip with mounting fluid. Read stain results and should not be used. under oil immersion.

*NOTE: Staining time can vary depending on the intensity desired for NOTE: The Xylene substitute, AmeriClear®, does not yield appropriate the final stain result. stain results and should not be used.

**NOTE: 100% Ethanol based reagent alcohol. *NOTE: Staining time can vary depending on the intensity desired for the final stain result. EXPECTED RESULTS Using the modified iron hematoxylin / acid-fast staining procedure with **NOTE: 100% Ethanol based reagent alcohol. organisms fixed in PROTO-FIX, PVA (Zn), or SAF, the cytoplasm of trophozoites and cysts will be tinged with violet. The nuclear chromatin, EXPECTED RESULTS chromatoid bodies, erythrocytes and bacteria stain dark purple to black. Using the modified iron hematoxylin / acid-fast staining procedure with Acid-fast organisms (i.e., Cryptosporidium parvum, Cyclospora organisms fixed in PROTO-FIX, PVA (Zn), or SAF, the cytoplasm of cayetanensis, and Isospora belli) will stain light to dark pink. Helminth trophozoites and cysts will be tinged with violet. The nuclear chromatin, eggs and larvae are difficult to distinguish with this staining procedure. chromatoid bodies, erythrocytes and bacteria stain dark purple to black.

L003561.E // CC19-021, Effective Date: 23 May 2019 // ©2019 Alpha-Tec Systems, Inc. All rights reserved. 2 [+1] 800.221.6058 United States Directions For Use for the following: [+1] 360.260.2779 International Iron Hematoxylin Stain Iron Hemotoxylin Mordant

Acid-fast organisms (i.e., Cryptosporidium parvum, Cyclospora cayetanensis, and Isospora belli) will stain light to dark pink. Helminth eggs and larvae are difficult to distinguish with this staining procedure.

LIMITATIONS OF PROCEDURES The Iron Hematoxylin Stain method is usually a trouble free procedure when used as directed. If problems occur, they will usually fall into one of the following categories.

STAIN RESULTS: egenerate forms will stain weakly. Unsatisfactory staining of the cytoplasm and the nucleus. CAUSE: Incomplete fixation of the smear resulting from failure to thoroughly emulsify the sample in the fixative, or failure to use enough fixative solution per sample. SOLUTION: Recollect the specimen following the directions in reference to the amount of specimen per fixative. Add enough specimen to meet the: “ADD SPECIMEN TO THIS LINE” mark on the label of the fixative tube. Mix the specimen well after adding it to the fixative.

STAIN RESULTS: Smears lack contrast. Smear appears cloudy (caused by incomplete penetration of 100% ethanol or xylene). CAUSE: Carryover of staining or decolorizing solutions from one step to another. Excessive staining will weaken or dilute the stain. SOLUTION: Change all solutions regularly to avoid staining clarity problems. Staining of over 30-40 slides (depending on the type of staining apparatus) will weaken or dilute the stain.

BIBLIOGRAPHY 1. Garcia, L.S. 2001. Diagnostic Medical Parasitology, 4th Ed. ASM Press, Washington, D.C. 2. Garcia, L.S. 2010. Clinical Microbiology Procedures Handbook, 3rd Ed. ASM Press, Washington, D.C. 3. Garcia, L.S. and L.R. Ash. 1979. Diagnostic Parasitology Clinical Laboratory Manual, 2nd Ed. pp. 19-24. The C.V. Mosby Co., St. Louis. 4. Melvin, D.M. and H.M. Brooke. 1982. Laboratory Procedures for the Diagnosis of Intestinal Parasites, 3rd Ed. US Department of Health, Education, and Welfare, Publication No. (CDC) 80-8282, pp. 111-131. Centers for Disease Control, Atlanta. 5. Price, D.L. 1994 Procedure Manual for the Diagnosis of Intestinal Parasites, CRC Press Inc., Cleveland, OH.

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: CELL-BOND®, ESC-1™, PRO-CLEAR™, PROTO-FIX® and QC1™ are trademarks of Alpha-Tec Systems, Inc., 1311 SE Cardinal Court, Suite 170, Vancouver, WA 98683 USA.

AmeriClear® is a trademark of Cardinal Health, McGaw Park, IL 60085, USA.

PRODUCT CODES 0003561 Iron Hematoxylin Stain, 1 L 0003562 Iron Hematoxylin Mordant, 1 L