Löffler's Staining Methods

LEFFLER'S STAINING METHODS, MEDIA.- 1. Gentian violet, or methyl violet. To 100 cm³ freshly prepared 1% or 2% carbolic water, add 10 cm³ of saturated alcoholic solution of gentian violet or methyl violet (6 B or BN). The staining power is increased by adding 1 cm³ of alcoholic solution of methylene blue for every 10 cm³ of the specified methyl violet solution. A widely used stain for staining bacteria.-2. L.'s modification of Gram's method. The object is stained in the specified solution of methyl or gentian violet for 10 min., washed in water, iodinated for 2 minutes in Lugol's solution, immersed for 1 min. in 5% aqueous solution of sulfuric or nitric acid (or for 10 sec. in 3% solution of hydrochloric acid), decolorized in absolute alcohol. Xylene. Canadian balsam. The method is used for staining bacteria in tissue sections.-3. Methylene blue. To 30 cm³ of saturated alcoholic solution of methylene blue, add 100 cm³ of 0.01% solution of KOH (for 100 cm³ distilled water, 1 cm³ of 1% solution of KOH). One of the common stains in bacteriology for staining bacteria in smears containing cellular elements; bacteria are stained more strongly than the background of cellular elements.-4. Staining of gonococci, a) Fixation of preparations for 40 min. in a mixture of alcohol and ether (aa); b) staining for 1 minute with gentle heating in a solution of 4 parts of a solution of 2.5 g borax and 1 g methylene blue in 100 cm³ distilled water, 1 part of polychrome methylene blue Unna, and 5 parts of 0.05% aqueous solution of Bromeosin extra B or extra A. G. (Hochst); c) washing with water; d) decolorization in a solution of 177 parts of 95° alcohol, 20 parts of 1% solution of brom eosin, and 3 parts of acetic acid; e) washing with water, drying.-Cells of pale pink color, nuclei-pale blue, gonococci-dark blue. 5. Staining of diphtheria bacilli and L. a) Fixation on a gentle flame; b) staining for 10 sec. in a solution of 4 parts of a solution of borax and methylene blue (2.5 g borax + 1 g methylene blue + 100 cm³ distilled water), 1 part polychrome methylene blue (Grübler), 5 parts of 0.05% solution of Bromeosin extra A. G. (Hochst); c) washing with water; d) decolorization for 10 sec. in a solution of a mixture of 500 cm³ of 25% acetic acid, in which 0.01 g Tropaeolin 00 is dissolved, and 500 cm³ of alcoholic solution of Bismarck brown (1 g Bismarck brown in 500 cm³ Alcohol, absolut.); e) washing with water, drying. Granules of diphtheria bacilli are stained black.- 6. Staining of flagella. Mordant: 10 cm³ of 20% aqueous solution of tannin is mixed with 5 cm³ of saturated cold solution of ferrous sulfate or ammonium alum; 1 cm³ of aqueous or alcoholic solution of fuchsin, methyl violet, or Wollschwarz is added.-s t a i n i n g. a) Fixation by three passes through the flame; b) mordant for */*-1 min. with heating until vapors appear (according to other authors, it is necessary to mordant 3-4 times for 10 sec., washing off the mordant with water in between); c) washing with a strong stream of water; d) washing with alcohol to remove sediment; e) staining for 3-4 min. with aniline water fuchsin with heating (the usual dye solution is alkalized by adding 1 cm³ of 1% solution of NaOH for every 100 cm³ of dye; alkali can be added in larger amounts-up to the beginning of turbidity of the solution); it is necessary to use a fresh solution; for staining, concentrated carbolic fuchsin or alcoholic water fuchsin can be used; f) washing with water, drying. The specified method is highly recommended for staining flagella of the coli-typhus group, large spirilla, Proteus, the anthrax bacillus group; for staining cholera and other vibrios, the method of Zettnow is more suitable. Löffler's method can be used for staining bacterial capsules.-7. Antiformin-chloroform method for detection of tubercle bacilli. Sputum is mixed with an equal amount of 50% antiformin, boiled; to 10 cm³ of the solution, 1.5 cm³ of 10% solution of chloroform in alcohol is added, shaken vigorously, centrifuged for 15 minutes. The layer formed between chloroform and antiformin is taken with a Pasteur pipette, mixed with a small amount of protein solution on a slide, fixed and stained.-8. Staining of mallein bacilli. a) Fixation by three passes of the preparation through the flame; b) staining for 5 min. in alkaline solution of methylene blue; c) decolorization by immersion in 1% acetic acid, which has the color of white wine from the addition of aqueous solution of Tropaeolin; d) washing with distilled water; drying. From the action of tropaeolin, there is decolorization of the cell plasma and partly of the nuclei, while bacteria are not decolorized. 9. Stained media for differential diagnosis of the coli-typhus group.-L i q u i d media. a) Medium for B. typhi: in 800 cm³ distilled water, dissolve 40 g of dry peptone and 20 g of grape sugar with heating; add 30 cm³ of normal solution of KOH and 200 cm³ water, in which 20 g of nutrose is previously dissolved; then 100 g of milk sugar is added. The medium is poured into flasks of 100 cm³ capacity and sterilized for 3 consecutive days by flowing steam for 20 min. Before use, 1 cm³ of sterilized 0.2% aqueous solution of malachite green is added to 100 cm³ of the cooled medium, after which the medium is sterilely poured in 4-5 cm³ portions into test tubes, b) Medium for B. paratyphi is prepared as the previous one, but without the addition of grape sugar. In the typhoid medium (a), B. enteritidis, B. paratyphi A and B, and coli bacteria cause fermentation within 24 hours, noticeable by the appearance of a green foamy ring in the upper layer of the medium; nutrose precipitates in the form of dirty flakes. B. typhi causes complete coagulation, as in milk; above the coagulum-transparent green liquid. In the paratyphoid medium (b), Bact. coli causes fermentation with foam formation, B. typhi and paratyphi A do not change the medium, B. paratyphi B and B. enteritidis do not cause fermentation, but slowly decolorize the medium.-M o d i f i c a t i o N S (Löffler) of the medium. 1. To 100 cm³ of paratyphoid medium (b), add 1 cm³ of 0.2% solution of safranin (Grübler). The medium then acquires a gray-violet color. Bact. coli causes fermentation, B. typhi and B. paratyphi A give a darker color to the medium, from B. paratyphi B the medium acquires a bright light red color (reduction of malachite green). 2. To both media (a and b), for every 100 cm³, add 1 cm³ of 0.2% solution of safranin, 2 cm³ of 0.2% solution of malachite green, and 3 cm³ of 1% solution of Reinblau. In solution (a) with grape sugar, after 24 hours, B. typhi causes blue-violet coloring and precipitation of blue sediment; B. paratyphi A-weak fermentation and turbidity of the medium; B. paratyphi B-strong fermentation and blue-red coloring of the medium; B. Gartner's mouse typhoid, Bact. coli-strong fermentation and blue-red coloring; Bact. paracoli-formation of blue sediment. 3. For every 100 cm³ of both media (a and b), add 1 cm³ of 0.2% solution of safranin and 3 cm³ of 1% solution of Reinblau. B. typhi and paratyphi A in medium a give formation of blue sediment, medium b is colored dark violet; after 36 hours, B. typhi gives strawberry-red coloring, B. paratyphi A-violet-blue; B. paratyphi B, B. Gartner's mouse typhoid cause fermentation and precipitation of blue sediment in medium a and strawberry-red in medium b; Bact. coli causes the same change in medium a, fermentation, coloring of the medium and formation of violet sediment in medium b. Solid media. The media described below, stained with malachite green, have the ability to inhibit the growth of Bact. coli and a number of alkali-forming bacteria; to some extent, there is also inhibition of the growth of B. typhi, however, after 24 hours, the latter gives on agar the formation of small transparent colonies, which after 2-4 days turn into large colonies coloring the medium yellowish.-M a l a c h i t e g r e e n-a g a r. To 5 liters of broth (1 kg of beef or horse meat in 5 liters of water), add 150 g of crushed agar, the mixture is boiled for ¹/₂ hour. If the agar does not dissolve well, 35 cm³ of normal HCl is added, which after dissolution is neutralized by 35 cm³ of normal solution of NaOH. Further, the medium is neutralized with soda to litmus, then again alkalized by adding 25 cm³ of normal solution of soda and brought to a boil. Into the boiling liquid, 500 cm³ of 10% solution of nutrose is added; after new boiling, the hot liquid is poured into half-liter flasks, and the medium is sterilized for 2 consecutive days for 2 hours in Koch's apparatus. The transparent agar is decanted from the sediment formed at the bottom.

To every 100 cm³ of melted to a liquid state and cooled to 50° agar, 1.5 cm³ of a 0.2% solution of chemically pure malachite green on sterilized water is added aseptically. The colored agar is poured into Petri dishes. - Malachite green-gelatin. 1¹/₂ kg of pure ground beef is poured with 5 l of water, 750 g (15%) of gelatin, 50 g (1%) of dry Witte peptone, and 25 g (0.5%) of NaCl are added. The mixture is slowly heated until the gelatin is completely dissolved, after which it is boiled for ³/₄ hour. The hot liquid is neutralized with soda according to litmus. It is again brought to a boil, then filtered. A clear golden-yellow liquid is obtained. To every 100 cm³ of such gelatin, 3 cm³ of normal phosphoric acid and 2 cm³ of a 2% solution of malachite green on sterilized water are added. - Malachite green-Reinblau-safranin-agar. 1 kg of ground beef is poured with 5 l of water, boiled for 1 hour, filtered, and brought with water to a volume of 5 l. 150 g (3%) of ground agar is added to the broth, after which the mixture is heated until it dissolves. The medium is neutralized with a saturated soda solution according to litmus (sensitive litmus paper should give: blue-dark violet spot, and red-pale blue). After neutralization, 25 cm³ of normal soda solution is added. The medium is again brought to a boil, to it is added 50 g of nutrose, slowly poured into 500 cm³ of water at t° 70°. After new boiling, the medium is poured into half-liter flasks of neutral glass and sterilized for 2 days in a row for 2 hours in Koch's apparatus; left in the apparatus until cooled. A precipitate forms at the bottom of the flasks, the agar above it should be transparent and have a light yellowish-white color. The appearance of a brown color indicates excessive addition of alkali, which suppresses the growth of typhoid bacteria. Before use, the flask with agar is heated for one hour in Koch's apparatus; to every 100 cm³ of transparent agar obtained by decanting and cooled to 45°, aseptically is added: a) 3 cm³ of sterilized and filtered ox bile; b) 1 cm³ of 0.2% sterile aqueous solution of safranin (Gribler); c) 3 cm³ of 1% sterile aqueous solution of Reinblau (double strength) (Hochst); d) 3-4 cm³ of 0.2% sterile aqueous solution of malachite green. After thorough mixing, the medium is poured into Petri dishes. The ready medium has a blue color with a blue-violet tint in reflected light. 10. Serum medium. The elective serum medium proposed by L. for diphtheria bacilli is prepared from 3 parts of calf or sheep serum and 1 part of neutral 1% sugar broth. The indicated mixture is poured into test tubes, which are placed in an inclined position, or Petri dishes and heated in a coagulation apparatus for 2 hours 3 days in a row at t° 90-95°. The mixture coagulates from heating, and a solid nutrient medium is formed. Heating must be done slowly to avoid the formation of foam or bubbles on the medium, in no case allowing the water in the apparatus to boil. Serum containing blood pigment colors the medium a grayish color instead of the required ivory color. The use of alkaline sugar broth makes the medium soft and gives it a brown color.