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| Hematology Urology Blood Bank Microbiology Cytology/Histology Miscellaneous |
METHANOL FIXATIVE For use as a fixative prior to staining bloody specimen material or blood culture supematant fluid. Methanol preserves the morphology of red blood cells as well as bacteria. RECOMMENDED PROCEDURE: Place material to be stained on a clean glass slide. Flood slide with methanol fixative for 1 minute. Drain without rinsing and allow slide to air dry prior to staining.
CONTENTS:
Methanol GRAM STAIN KIT Solution I - Crystal Violet AVAILABLE IN VARIOUS COUNTERSTAINS AS FOLLOWS: Safranin - Routinely used for gram negative bacteria. Basic Fuchsin - More intense overall staining to clearly identify weak gram-negative bacteria and also aids in the detection of Bordatella. Carbol Fuchsin - A counterstain of greater overall intensity to easily identify Legionella. Also available with various decolorizers to suit individual preference of technique. Convenient flip-top cap for 250 ml and 950 ml sizes. RECOMMENDED PROCEDURE: Cover fixed smear with Sol. I for 1 minute and rinse gently with distilled water. Apply Sol. II for 1 minute and rinse gently with distilled water. Decolorize with Sol. III, Sol. IIIA, or Sol. IIIB until the purple color no longer runs off the slide, (from 2 - 20 seconds depending upon decolorizer and thickness of smear) and rinse with distilled water. Counterstain with Sol. IV, Sol. IVA or Sol. IVB for 1 minute and rinse with distilled water. Allow to dry and examine under oil immersion. NOTE: For a faster decolorizer, use Sol.IIIB - Acetone-Alcohol. For a more intense counterstain, we recommend Sol. IVA - Basic Fuchsin or Sol. IVB - Carbol Fuchsin. RESULTS: Gram-positive bacteria stain purple to blue black. Gram-negative bacteria stain pink to red.
CONTENTS: Crystal Violet, Ethanol, Potassium Iodide, Iodine, Acetone, Safranin, Basic Fuchsin, Phenol These solutions are made from certified
dyes (when applicable). GRAM STAIN KIT Solution IA - Crystal Violet RECOMMENDED PROCEDURE FOR OPTIMUM RESUTLS: 1. Cover the fixed slide with Solution IA and then add 5 drops of Sodium Bicarbonate and blow on slide to mix. Let stand for 15 seconds. Continue staining as usual for Gram Stain. Wash off gently with distilled water. NOTE: Crystal Violet is used because it is more stable than Gentian Violet. 2. Apply Solution II, (Kopeloff's Iodine modification) for 1 minute and wash off gently with distilled water. 3. Decolorize with Solution III, Acetone Alcohol until the purple color no longer runs off the slide. (From 2 to 20 seconds depending upon thickness of specimen) Wash with distilled water. 4. Cover slide with Solution IV, Safranin for 1 minute. Wash with distilled water and allow to dry. RESULTS: Gram-positive bacteria stain purple to blue-black. Gram-negative bacterial stain pink to red.
CONTENTS: Crystal Violet, Ethanol, Potassium Iodide, Iodine, Acetone, Safranin, Basic Fuchsin, Phenol Crystal Violet, Sodium Bicarbonate, Sodium Hydroxide, Potassium Iodide, Iodine, Ethanol, Acetone, Safranin These solutions are made from certified
dyes (when applicable). GRAM STAIN KIT Solution IA - Crystal Violet RECOMMENDED PROCEDURE FOR OPTIMUM RESUTLS: 1. Cover the fixed slide with Solution IA and then add 5 drops of Sodium Bicarbonate and blow on slide to mix. Let stand for 15 seconds. Continue staining as usual for Gram Stain. Wash off gently with distilled water. NOTE: Crystal Violet is used because it is more stable than Gentian Violet. 2. Apply Solution II, (Kopeloff's Iodine modification) for 1 minute and wash off gently with distilled water. 3. Decolorize with Solution III, Acetone Alcohol until the purple color no longer runs off the slide. (From 2 to 20 seconds depending upon thickness of specimen) Wash with distilled water. 4. Cover slide with Solution IV, Basic Fuchsin for 1 minute. Wash with distilled water and allow to dry. RESULTS: Gram-positive bacteria stain purple to blue-black. Gram-negative bacterial stain pink to red.
CONTENTS: Crystal Violet, Ethanol, Potassium Iodide, Iodine, Acetone, Safranin, Basic Fuchsin, Phenol Crystal Violet, Sodium Bicarbonate, Sodium Hydroxide, Potassium Iodide, Iodine, Ethanol, Acetone, Basic Fuchsin These solutions are made from certified
dyes (when applicable). CYCLOSPORA STAIN KIT Solution I - Carbol Fuchsin RECOMMENDED PROCEDURE: Specimen may be prepared from fresh material or preserved in 10% buffered formalin (Product # 9700). Formalin-preserved Specimens: Mix stool specimen thoroughly and filter an adequate amount through cheesecloth into a centrifuge tube. Centrifuge for 2 minutes and decant supernatant. Smear 1 to 2 drops of specimen on a glass slide (either concentrate or original specimen). Heat fix on slide warmer until dry (about 5 minutes). Fresh Stool Specimens: Prepare smear utilizing the same procedure as for a formalin-fixed specimen. Fix slide in absolute methanol - 30 seconds. Flood slide with Sol. I for 1 minute. (Enhanced staining can be obtained by increasing time or applying heat). Rinse with distilled water and drain. Decolorize with Sol. II for 2 minutes. Rinse with distilled water and drain. Counterstain with Sol. III for 2 minutes. Rinse briefly with distilled water and drain. Dry on slide warmer until dry and mount. RESULTS: Cyclospora oocysts stain light pink to deep red measuring 8-10 microns.
CONTENTS: Basic Fuchsin, Ethanol, Phenol, Sulfuric Acid, Malachite Green These solutions are made from certified dyes (when
applicable). CRYPTOSPORIDIUM STAIN KIT Solution I - Carbol Fuchsin This technique aids in differentiating Crytosporidium oocysts from yeast cells. RECOMMENDED PROCEDURE: Specimen may be prepared from fresh material or preserved in 10% buffered formalin (Product # 9700). Formalin-preserved Specimens: Mix stool suspension thoroughly and filter an adequate amount through cheesecloth into a centrifuge tube. Centrifuge for 2 minutes, decant supernatant and add saline to the remaining sediment. Centrifuge again and decant saline. Smears may be prepared from upper layer of sediment. Material should be spread on a clean glass slide and allowed to air dry. Fix fresh specimens in methanol and formalized specimens with heat. Flood smear with Sol. I for 1 - 2 minutes. Rinse gently with water. Decolorize with Sol. II and rinse again with water. Counterstain with Sol. III for 30 - 60 seconds. Rinse with water and allow to dry. NOTE: Decolorization and counterstaining times may vary according to thickness of specimen. RESULTS: Cryptosporidium oocysts - light pink to deep red; yeast cells - green to blue-purple.
CONTENTS: Basic Fuchsin, Ethanol, Phenol, Glacial Acetic Acid, HCl, Light Green SF These solutions are made from certified
dyes (when applicable). WAYSON STAIN Yersinia Pestis is a gram-negative bacillus with rounded ends occurring in pairs or very short chains. Bipolar staining ("safety pin" appearance) and vacuolated involutions forms are characteristic for the organism. Yersinia Pestis can be identified using a Gram Stain or Loeffler's Methylene Blue. Bipolar staining is characteristic and is best visualized with Wayson Stain. RECOMMENDED PROCEDURE: Prepared slides are air dried and heat fixed. Stain slides for 10 - 20 seconds. Rinse with distilled water and blot dry. RESULTS: Yersinia Pestis organisms stain dark blue, leukocytes stain light blue or purple, and the background stains a light blue.
CONTENTS: Methylene Blue, Basic Fuchsin, Phenol, Ethanol This solution is made from certified dyes. SPUTUM DIGESTANT A digestant for sputum, gastric washings, etc., in preparing for acid-fast staining or culture. RECOMMENDED PROCEDURE: Mix equal amounts of sputum and sputum digestant. Mix well and incubate in 37oC water bath for 30 minutes with occasional shaking. With 2.5 normal HCl (Product # 9789), neutralize digested sputum to red end point. On an average, 1 - 3 ml may be needed. Flocculation will occur after 30 seconds. Centrifuge for 5 minutes then remove and discard supernatant fluid. Smears for acid-fast staining may be made directly or the diluted sediment may be used for culture. All normal flora is rendered non-viable.
CONTENTS: Sodium Hydroxide, Potassium Alum, Methyl Red. FOR IN VITRO DIAGNOSTIC USE ONLY. ACID-FAST STAIN KIT Solution I - Carbol Fuchsin
Solution II - Acid Alcohol RECOMMENDED PROCEDURE: Fix slide gently with heat. Flood slide with Sol. I and stain for 2 minutes or longer depending on thickness of smear. Rinse with water. Decolorize slide in Sol. II. Counter stain with Sol. III or Sol. IIIA for 2 minutes. Wash with water and allow to dry. RESULTS: Acid-fast bacilli stain red; all other bacteria stain blue with Sol. III, green with Sol. IIIA.
CONTENTS: Phenol, Basic Fuchsin, Ethanol, HCl, Methylene Blue, Malachite Green These
solutions are made from certified dyes (when applicable). ACID-FAST STAIN
KIT Solution I - Carbol Fuchsin The nocardiae, because of unusual long-chain fatty acids in their cell walls, can retain carbol fuchsin dye with a decolorizer of 1% sulfuric acid in water, whereas other aerobic branching bacilli cannot. RECOMMENDED PROCEDURE: Organisms to be stained should be emulsified in a drop of distilled water on a glass slide. A known positive control and a known negative control should be stained along with the unknown strain. Allow to air dry and heat fix. Flood smear with Sol. I for 3 minutes. Wash with water. Decolorize briefly for 5 - 10 seconds with Sol. II. Counterstain with Sol. III for 30 seconds. Wash with water and allow to dry. NOTE: Due to the organisms weak or partial acid-fastness, do not exceed recommended decolorizing time. RESULTS: Partially acid-fast organisms show reddish to purple filaments; non-acid-fast organisms stain blue.
CONTENTS: Basic Fuchsin, Ethanol, Methylene Blue, Phenol, Sulfuric Acid These solutions are made from certified
dyes (when applicable). PPLO STAIN Mycoplasma, small bacteria without cell walls, are destroyed by the usual methods of smear preparation and heat fixation. On solid culture medium, they form small colonies and are best stained through direct staining of these young colonies. RECOMMENDED PROCEDURE: On a clean, grease-free coverglass, smear a thin even film of stain and allow to dry. With a sterile scalpel, cut a block of agar from a fresh culture of the organism. Place on a clean glass slide with colonies facing upward. Apply coverglass, stain side down, gently on the agar block without rubbing. Seal with a melted mixture of paraffin and 10% petrolatum. Examine the surface of the agar for stained colonies under low-power objective. RESULTS: Mycoplasma colonies stain distinctly with dense blue centers and light blue peripheries.
CONTENTS: Methylene Blue, Azure II, Maltose, Sodium Carbonate This solution
is made from certified dyes. MODIFIED TRICHROME
STAIN KIT Solution I - Formalin (10%) This stain method was designed as a simple, non-invasive technique for the rapid detection of microsporidia spores in fecal material by light microscopy. The chromotrope based stain is a modification of the trichrome stain for intestinal protozoa. RECOMMENDED PROCEDURE: Specimens for light microscopial examination should be prepared from a suspension of unconcentrated liquid stool (10 ul) in Sol. I (1:3 ratio). Smears are prepared directly and thinly spread on a glass slide covering a 45 x 25 mm area. Fix smears in Sol. II for 5 minutes. Stain smears with Sol. III for 90 minutes. Rinse slides in Sol. IV for 10 seconds. Rinse briefly in Sol. V to remove acid. Dehydrate in Sol. V for 5 minutes, Sol. VI for 10 minutes and Sol. VII for 10 minutes. Read microscopically under oil immersion lens (magnification x 1000). RESULTS: Enterocytozoon bienuesi spores appear ovoid and refractile at approximately 1.5 by 0.90 um. The spore wall of properly stained smears should appear a bright pinkish red. Most bacteria and background debris stain a faint green.
CONTENTS: Chromotrope 2R, Fast Green FCF, Phosphotungstic Acid, Glacial Acetic Acid. These
solutions are made from certified dyes (when applicable). MODIFIED IRON HEMATOXYLIN STAIN SOLUTIONS Solution IA - Alcoholic
Hematoxylin A solution used in the Modified Iron Hematoxylin Stain Procedure that can incorporate a Carbol Fuchsin Step to screen for acid-fast organisms and other intestinal parasites. RECOMMENDED PROCEDURE: Working Solution: Mix equal parts of Solution 1A and Solution 1B. Recommended staining time is 8 minutes. RESULTS: Background material should be stained various shades of gray-blue and the protozoa, with medium blue cytoplasm and dark blue nuclei.
CONTENTS: Hematoxylin, Ethanol, Hydrochloric Acid, Ferrous Ammonium Sulfate, Ferric Ammonium Sulfate. This solution
is made from certified dyes. TRICHROME STAIN
KIT Solution I - PVA Solution (5%) NOTE: THIS STAIN IS NOT TO BE CONFUSED WITH GOMORI’S TRICHROME STAIN USED FOR HISTOLOGY. IT IS A RECOMMENDED METHOD BY THE ASCP. "MANUAL-LABORATORY DIAGNOSIS OF INTESTINAL PARASITIC INFECTION". A simple and quick technique for staining intestinal protozoa based on the principle of the attraction that these parasites show for the Chromotrope 2R dyes. PVA Solution, when mixed with liquid stool specimens, (3 - 4 drops PVA with 1 - 2 drops of fecal material on a slide) provides excellent adhesion of specimen to slides, if allowed to dry several hours at 35oC or overnight at room temperature. Schaudinn fixative will properly fix and hold fresh stool specimens that have been thinly spread on a slide. (5 minutes at 50oC or 1 hour at room temperature) Smears may be left in fixative for several hours or 1 - 2 days without harm. PVA fixative (a combination of PVA Solution and Schaudinn’s fixative) will adhere as well as preserve organisms and prevent their loss during staining. One distinct advantage of PVA fixative is that it makes possible the successful staining of organisms in liquid specimens. RECOMMENDED PROCEDURE: STAINING TECHNIQUE WITH PVA FILMS: PVA-fixed smears should be allowed to dry overnight at 37o degrees C prior to staining. Sol. II - 10 - 20 minutes. Sol. III - 3 - 5 minutes, then repeat. Sol. IV - 8 minutes. Sol. V - 10 - 20 seconds. Rinse with Sol. VI to remove acid. Sol. VI - 5 minutes. Sol. VII - 5 - 10 minutes. Sol. VIII - 10 minutes. Mount with coverslip. STAINING TECHNIQUE WITH FRESH SPECIMENS: Schaudinn’s fixative - 5 minutes at 50oC or 1 hour at room temperature. Sol. II - 1 minute. Sol. III - 1 minute, then repeat. Sol. IV - 8 minutes. Sol. V - 3 - 10 seconds. Sol. VI - rinse briefly, 2 changes. Sol. VII - 1 minute.Sol. VIII - l.- 3 minutes. Mount with coverslip. NOTE: CHANGE ALCOHOLS OFTEN. RESULTS: The cytoplasm of thoroughly-fixed and well-stained cysts and trophozoites are blue-green tinged with purple. The nuclear chromatin, chromatoid bodies, ingested red cells, and bacteria stain red or purplish red. Other ingested particles, such as yeast or molds generally stain green, but variations frequently occur in the color reaction of ingested particles.Other ingested particles, such as yeasts or molds, generally stain green, but variations in color frequently occur. Background material usually stains green, and a color contrast with the protozoa results.
CONTENTS: PVA Powder, Mercuric Chloride, Iodine, Ethanol, Chromotrope 2R, Light Green SF, Fast Green FCF, Phosphotungstic Acid, Acetic Acid (Glacial), Xylene, Pheno2 These solutions are made from certified dyes (when
applicable). CALCOFLUOR WHITE
STAIN Calcofluor white, a nonspecific fluorochrome, will bind to the cell walls of certain fungi allowing for the direct microscopic observation of fungi in wet preparations. RECOMMENDED PROCEDURE: Add a drop of stain to specimen on clean glass slide and coverslip. Place slide face down on paper toweling and push down gently allowing excess fluid to flow out of the edges of the coverslip on to the paper towels. Examine slide under UV light. RESULTS: Fungi elements fluoresce blue-white or green depending on filter system used i.e., a G-365 excitation filter and an LP 420 barrier filter produce light of blue-white wavelength, whereas, a K530 excitation filter and a BG 12 barrier filter produce light of green wavelength.
CONTENTS: Calcofluor White, Evans Blue. This product is made from certified dye. LACTOPHENOL
COTTON BLUE STAIN RECOMMENDED PROCEDURE: Mix culture material with stain (loopful) and coverslip. Lactic acid acts as a preservative for fungi, killed by phenol and stained by cotton blue. If bubbles are present, heat gently over flame. RESULTS: Fungal elements are stained a deep blue; background is pale blue.
CONTENTS: Phenol, Lactic Acid, Glycerol, Aniline Blue This solution is made from certified dyes. ALBERT’S
STAIN SOLUTION KIT Albert’s Stain Solution I This method differentiates volutin granules in the cells, and reveals the barred staining patterns of the cytoplasmic region. RECOMMENDED PROCEDURE: Stain heat-fixed smear with Sol. I for 3 - 5 minutes. Wash in water, blot and air dry. Stain with Sol. II for 1 minute. Wash again, blot and air dry. RESULTS: Volutin granules - dark blue or black; cytoplasm-light green; barred patterns - dark green.
CONTENTS: Toluidine Blue, Malachite Green, Ethanol, Acetic Acid, Iodine, Potassium Iodide. These solutions are made from
certified dyes (when applicable). MIF STAIN
AND FIXATIVE KIT Solution I - MF Stock Solution Stool preservation and staining can be accomplished simultaneously with this kit. When used as a stain, direct smear identification of intestinal protozoa and of helminth eggs may be performed immediately. As a preservative, laboratory diagnosis can be delayed without deterioration of organisms. RECOMMENDED PROCEDURE: Mix 2.35 ml of Sol. I with 0.15 ml of Sol. II. This is mixed with approximately 0.25 grams of feces (about twice the size of a pea) in a well-stoppered tube. To examine, use a medicine dropper to draw off a drop of the mixture from the layer of sediment. RESULTS: Trophozoites and cysts should stain an eosin color.
CONTENTS: Glycerol, Formaldehyde, Tincture Merthiolate, Iodine, Potassium Iodide FOR IN VITRO DIAGNOSTIC USE ONLY. SPORE
STAIN KIT (RECOMMENDED FOR STAINING BACILLUS SPECIES ANTHRACIS, CEREUS, ETC.) Solution I - Malachite Green (5%) RECOMMENDED PROCEDURE: Fix smear with heat. Apply Solution I - Malachite Green, and heat until steam rises. Continue for 3 to 6 minutes. Rinse under running tap water. Counterstain with Soluton II - Safranin for 30 seconds. Wash very lightly with water, blot dry, and examine. RESULTS: Spores are seen as green spherules in red-stained rods or with attached red-stained debris.
CONTENTS: Malachite Green, Safranin These
solutions are made from certified dyes (when applicable). SPORE STAIN KIT Solution I - Basic Carbol Fuchsin RECOMMENDED PROCEDURE: Heat fix smear. Apply Sol. I and heat over flame until steam rises. Continue for 5 minutes. Decolorize with Sol. II until smear assumes a light pink color. Counterstain with Sol. III or Sol. IlIA for 3 minutes. Wash with water and allow to dry. RESULTS: Bacteria - blue or green depending upon counter stain; spores-red.
CONTENTS: Basic Fuchsin, Phenol, Ethanol, Acetic Acid, Methylene Blue, Malachite Green These solutions are
made from certified dyes (when applicable). LUGOL’S IODINE SOLUTION Used primarily for the observation of protozoan cysts in wet mount preparations of fecal specimens. RESULTS: Iodine enhances the nuclear structure and stains glycogen masses dark brown.
CONTENTS: Iodine, Potassium Iodide FOR IN VITRO DIAGNOSTIC USE ONLY. CAPSULE
STAIN KIT Solution I - Aqueous Crystal Violet
(1%) RECOMMENDED PROCEDURE: Grow organisms in ascitic fluid or serum medium, or mix a drop of serum with specimen and from this mixture, prepare smear. Air dry. Cover smear with Sol. for 2 minutes. Wash off the stain with Sol. II. The amount of washing must be determined empirically. RESULTS: Capsules appear as faint blue halos around dark blue to purple cells.
CONTENTS: Crystal Violet, Copper Sulfate This solution
is made from certified dyes (when applicable). EOSINOPHIL STAIN KIT Solution I - Eosin Y Used in staining eosinophils from secretions of the nasal passage, sinuses, bronchi and urinary sediment. RECOMMENDED PROCEDURE: Make thin smears and heat fix or air dry. Apply Sol. I for 30 seconds. Add Sol. II to cover slide. Mix by blowing gently on slide. Allow to stain 3 - 5 minutes. Rinse with distilled water and drain completely or blot dry. Cover slide with Sol. III and immediately rinse with water. Drain and allow to dry. RESULTS: Eosinophils stain with red cytoplasm and bright red granules; nucleus stains pale blue.
CONTENTS: Eosin Y, Methanol, Buffer Salts, Methylene Blue These
solutions are made from certified dyes (when applicable). FLUORESCENT
STAIN KIT Solution I - Auramine Phenol This technique uses a phenolated fluorescent dye in place of carbol fuchsin. RECOMMENDED PROCEDURE: Stain heat-fixed smears 2 - 3 minutes in Sol. I. Wash in tap water. Destain 3 - 5 minutes in Sol. II. Wash in tap water. Flood smear with Sol. III for 2 - 4 minutes. Wash in tap water. Allow to dry. RESULTS: Acid-fast organisms should appear fluoresced against a dark background.
CONTENTS: Auramine O, Liquid Phenol, Ethanol, HCl, Potassium Permanganate These solutions are made from
certified dyes (when applicable). LEIFSON’S BACTERIAL FLAGELLA STAIN KIT Solution I - Basic Fuchsin, (1.2%)
Alcoholic Bacterial flagella are below the visual limit in size, approximately 0.1 to 0.3u in diameter. Flagella stains increase the width of these delicate appendages by forming a precipitate at the surface of the flagella making them visible with the optical microscope. The preparation may be used to establish polar or lateral distribution of flagella on a bacterial cell. RECOMMENDED PROCEDURE: Preparation of Slides: Slides must be clean and grease free. To assure this, soak slides for 4 days in a slide cleaning solution (Product # 6981). Thoroughly wash slides in distilled water and allow to dry. Working Solution: Mix equal parts of each solution (I, II, III) in a glass vessel and tightly stopper. Allow to stand 2 hours prior to use. NOTE: Working stain solution will stain flagella satisfactorily for only 2 - 3 days; therefore, only prepare as needed. This solution precipitates rapidly at room temperature. Use only the clear supematant for staining. From a centrifuged 24 hour broth culture of a motile species of bacteria, a loopful of sediment is placed at the end of the slide. Tilt the slide allowing the liquid to run slowly to the other end and air dry. Quickly add 1 ml of stain mixture. Allow to stand 5 - 15 minutes at room temperature. When precipitate forms over the entire area of the slide, carefully wash off by flooding with water. Counterstain with 1:l0 dilution of Sol. IV for 1 minute. Wash and drain dry. RESULTS: Flagella stain bright red.
CONTENTS: Basic Fuchsin, Ethanol, Tannic Acid, Sodium Chloride, Methylene Blue, HCl These
solutions are made from certified dyes (when applicable). ZIEHL-
NEELSEN STAIN KIT Solution I - Carbol Fuchsin RECOMMENDED PROCEDURE: Flood slide with Sol. I and heat until steam rises. Use low or intermittent heat to maintain steam for 3 - 5 minutes. After cooling, wash briefly with water and decolorize with Sol. II. Wash with water and counterstain with Sol. III for 20 - 30 seconds. Rinse with water, allow to dry and examine. RESULTS: Acid-fast organisms stain red; other organisms and material stain blue.
CONTENTS: Basic Fuchsin, Phenol, Ethanol, HCl, Methylene Blue These
solutions are made from certified dyes (when applicable). CHLAMYDIAL STAIN KIT Solution I - Giemsa Stain RECOMMENDED PROCEDURE: Giemsa Buffer Solution: 1 part of Sol. I to 40 or 50 parts of Sol. II. (If a darker stain is desired, increase ratio of stain to buffer.) Place fixed smear into Giemsa Buffer Sol. for 1 hour. (Time may vary according to intensity of stain desired.) NOTE: Giemsa Buffer Sol. should be freshly made. Slide is then rinsed rapidly in 95% ethanol to remove excess dye and to enhance differentiation. Iodine Method: Place fixed smear into Sol. III for 3 - 5 minutes. Slide is examined as a wet mount. NOTE: The slide may be decolorized with methyl alcohol and restained with Giemsa stain. RESULTS: Giemsa Stain: The elementary bodies stain a reddish purple. The initial bodies are more basophilic, staining bluish as do bacteria. Iodine Stain: The matrix of inclusions may appear as a reddish-brown mass recognizable under low power. Check with higher objective to confirm.
CONTENTS: Giemsa Stain, Methanol, Buffer Salts, Iodine, Potassium Iodide These
solutions are made from certified dyes (when applicable). PNEUMOCYSTIS
CARINII STAIN KIT Solution I - Toluidine Blue 0 Stain Pneumocystis carinii is an opportunist which causes a diffuse interstitial pneumonia in patients with impaired immune systems. The disease itself can be classified as epidemic or sporadic; the latter occurring in patients who have an underlying immunosuppression. In this procedure, a sulfation reagent of glacial acetic acid and sulfuric acid (not included) is used for the removal of background material. This allows the Pneumocystis carinii cysts to be visualized more easily after staining with Toluidine Blue 0. Diagnosis of Pneumocystis carinii pneumonia can frequently be made on bronchoalveolar lavage (BAL) or on touch preparations of pulmonary tissue (open lung biopsies and transbronchial biopsies). RECOMMENDED PROCEDURES: BAL Specimens: Using a 50 ml tube, centrifuge for 15 minutes at 2000 x g. Aspirate all but the bottom 5 ml of supernatant. With a Pasteur pipette, aspirate the sediment plus approximately 1 ml of the remaining 5 ml of fluid. Transfer material to a 15 ml centrifuge tube, gently mix and prepare smear by spreading a drop over a 1 cmm area. If concentrated specimen is very thick or mucoid, spread over entire slide with care being taken not to make the smear too thick. Dry the slides on a heating block at 50 - 55oC. Allow to cool before staining. Touch Preparations: When dry, process in same manner as slides of BAL. Preparation of Sulfation Reagent: In a dry coplin jar submerged in a plastic tub filled with cool water (not below 10oC), mix 45 ml of glacial acetic acid with 15 ml of concentrated sulfuric acid and stir gently with glass rod. Carefully place smears in reagent for 10 minutes. Mix immediately and again after 5 minutes. Gently rinse with water for 10 minutes. Drain excess water and stain with Sol. I for 3 - 5 minutes. Rinse with Sol. II followed by Sol. III for 10 seconds each. Clear in Sol. IV, two changes each for 10 seconds and mount. RESULTS: The cyst forms appear as lavender structures (cup-shaped) approximately 5 urn in diameter. The cyst outline is distinct, and the internal region stains uniformly. NOTE: A negative bronchoalveolar lavage does not rule out an infection with Pneumocystis carinii. Other diagnostic procedures may be necessary.
CONTENTS: Toluidine Blue O, HCl, Ethanol, Xylene These
solutions are made from certified dyes (when applicable). GRAM-WEIGART
STAIN KIT Solution I - Eosin Y, (1%) Aqueous This stain kit is useful in demonstrating Pneumocystis carinii in smears of sputum, transtracheal aspirates, and bronchial washings, however, lung biopsy specimens obtained by percutaneous needle aspiration provide a better opportunity for diagnosis. Tissue sections and imprints may also be used. Prior to staining, imprints and smears must be air-dried and fixed in methanol. RECOMMENDED PROCEDURE: Working Solution: Mix 10 ml of Sol. IIA with 90 ml of Sol. IIB. Stain air-dried smears or imprints in Sol. I for 3 - 5 minutes. Rinse with water. Stain in working solution for 5 - 10 minutes. Rinse in Sol. III for 5 - 10 minutes. Rinse in water and blot dry. Decolorize in Sol. IV and rinse in Sol. V. RESULTS: Pneumocystis appears blue to deep purple against a pink background.
CONTENTS: Eosin Y, Crystal Violet, Ethanol, Aniline Oil, Iodine, Potassium Iodide, Xylene These
solutions are made from certified dyes (when applicable. MACCHIAVELLO
STAIN KIT Solution I - Basic Fuchsin Rickettsiae and chlamydiae are considered to be small bacteria since they are prokaryotic cells possessing DNA and RNA and they reproduce by the process of binary fission. They differ from all other bacteria by being obligate intracellular parasites. Since they are gram negative, gram stain does not differentiate them from the cellular material with which they are usually associated. The Macchiavello stain is considered to be one of the best differential stains available. The mechanism of this staining procedure is based on the affinity of cationic (basic) dyes for rickettsial and chlamydial organisms. This is emphasized in the decolorization step where cellular material is decolorized faster than the microorganisms. RECOMMENDED PROCEDURE: Prepare a smear from tissue or yolk-sac material on a clean glass slide and allow to air dry. Lightly heat fix slide. Smears should be processed soon after preparation and not allowed to remain at room temperature unstained. Cover smear with Sol. I for 5 minutes. Drain off excess dye. Wash slide in water and then rinse briefly with Sol. II. Rinse smear in water again. Counterstain with Sol. III or Sol. IIIA for 10 seconds. Rinse again, blot dry and read microscopically. NOTE: Exposure to citric acid for more than a few seconds will decolorize the organisms staining them blue instead of red. RESULTS: In a properly prepared slide, rickettsiae and chlamydiae stain red, cellular material stains blue or green.
CONTENTS: Basic Fuchsin, Citric Acid, Methylene Blue, Malachite Green These
solutions are made from certified dyes (when applicable). |
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