Bacterial antacid staining can be applied to (1) staining bacteria that are not susceptible to other staining materials (2) morphological analysis of bacteria.

Operation method

Bacterial antacid staining

Principle

Mycobacterium tuberculosis is generally not easy to aniline dye coloring, if the temperature or prolong the dyeing time to make it coloring, and then 3% hydrochloric acid alcohol treatment is not easy to decolorization, after this staining, Mycobacterium tuberculosis and other mycobacteria are red, while non-acid-resistant bacteria and cellular impurities, etc. are blue.

Materials and Instruments

Sputum of tuberculosis patients
Antacid staining solution Alcohol hydrochloride Lue's Meconium Blue Stain
Microscope, slide, inoculation ring

Move

1. Take a clean bamboo skewer and dip the sputum into blood or purulent sputum and smear it on a clean, grease-free slide. The smear area is about the size of the thumb cap. Put the used bamboo stick into the empty flat dish and wait for autoclaving.

After the smear has dried naturally, one end of the slide is held by a clip and fixed by flame.

3, smear surface with a piece of filter paper to cover, and then add drops of carbolic acid red dye to the filter paper, so that the filter paper is completely wet by the dye, hold the slide on a small fire heating for a moment and then leave the flame, at this time you can see the slide on the steam. To be vapor disappears and then warmed, so repeated 3 to 4 times, about 5min, warming at any time to add the dye solution do not make the paper dry.

4, after the slide cooling, with the inoculation ring pick off the filter paper thrown into the dirt basin, rinse the slide with running water.

5, drop 3% hydrochloric acid alcohol decolorization, to smear uniform parts of the basic no red and then take off until, water rinse.

6、Drop and add Lv's beauty blue dye solution 30sec, water washing.

7、Absorb dry, microscopic examination.

4、Solution preparation

Zichl-Neelsen (Zichl-Neelsen) antacid staining solution

(1) Carbonic acid red solution: alkaline red 4g, dissolved in 95% alcohol 100ml, made into a saturated solution, and then take 10ml of the saturated solution and 5% carbonic acid solution 90ml mixing into.

(2)3% hydrochloric acid alcohol solution.

Concentrated hydrochloric acid 3 ml added to 95% alcohol 97 ml to become.

(3)Lue (Loeffer)'s Meilan staining solution

Loeffer's blue 2g, dissolved in 95% alcohol 100ml, made into a saturated solution. Then take 30ml of the saturated solution and 0.01% potassium hydroxide solution 100ml mix well.

Caveat

1. Avoid direct contact with biological samples during use to avoid infection.

2. Sterilize the samples after use and place them in a designated location for uniform disposal.

Common Problems

Acid-fast staining method Bacterial staining method created by F. Ehrlich in 1882 and improved by F. Ziehl. The most representative ones are the Ziehl-Neelsen staining method and the Ziehl-Gabbet staining method for M. tuberculosis. After staining with carbolic acid red, the color is separated by hydrochloric acid ethanol, and then contrast stained with American blue, that is, it is no longer decolorized and shows the red color of carbolic acid red.

Solution preparation

Zichl-Neelsen antacid staining solution

(1) Carbonic acid red solution: alkaline red 4g, dissolved in 95% alcohol 100ml, made into a saturated solution, and then take 10ml of the saturated solution and 5% carbonic acid solution 90ml mixing into.

(2)3% hydrochloric acid alcohol solution.

Concentrated hydrochloric acid 3 ml added to 95% alcohol 97 ml to become.

(3)Lue (Loeffer)'s Meilan staining solution

Loeffer's blue 2g, dissolved in 95% alcohol 100ml, made into a saturated solution. Then take 30ml of the saturated solution and 0.01% potassium hydroxide solution 100ml mix well.

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