Acid-fast stain 染色法 Acid-fastness is a physical property of some bacteria referring to their resistance to decolorization by acids during staining procedures.

Presentation on theme: "Acid-fast stain 染色法 Acid-fastness is a physical property of some bacteria referring to their resistance to decolorization by acids during staining procedures."— Presentation transcript:

1 Acid-fast stain 染色法 Acid-fastness is a physical property of some bacteria referring to their resistance to decolorization by acids during staining procedures

2 Purpose Some bacteria such as Mycobacterium species have special cell wall structure, which strongly retain dye and can not be destained by acidic ethanol.

3 Introduction/principle
Acid-fast organisms are difficult to characterize using standard microbiological techniques such as Gram staining, though they can be stained using concentrated dyes, particularly when the staining process is combined with heat. Once stained, these organisms resist the dilute acid and/or ethanol-based de-colorization procedures common in many staining protocols—hence the name acid-fast

4 All Mycobacterium species share a characteristic cell wall, thicker than in many other bacteria, which is hydrophobic, waxy, and rich in mycolic acids/mycolates. The cell wall consists of the hydrophobic mycolate layer and a peptidoglycan layer held together by a polysaccharide, arabinogalactan. The cell wall makes a substantial contribution to the hardiness of this genus.

5 Bacteria: Mycobacterium mucogenicum
Gram-positive, nonmotile, curved and acid-fast rods. The Latin prefix “myco—” means both fungus and wax; its use here relates to the “waxy” compounds in the cell wall. (漢字: 分歧) mucogenicum :from the organism's highly mucoid appearance.

6 Genus Mycobacterium contains several important human pathogenic species.
The most important pathogenic species is M. tuberculosis (MTB) (結核菌). Therefore, fast-acid stain is called TB stain in medical clinics. Leprosy (from the Greek lepi (λέπι), meaning scales on a fish), (痲瘋) or Hansen‘s disease (漢生(HD) is caused by M. leprae (identified by Norwigian Dr. Gerhard Henrik Armauer Hansen in 1873) and M. lepromatosis.

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9 Materials and Methods bacteria: Stain agents:
Mycobacterium mucogenicum (obtained from Dr. 陳文明) Escherichia coli Staphylococcus aureus as controls Stain agents: Carbolfuchsin (primary) Acid alcohol (decolor) Methylene blue (secondary)

10 methods Prepare slides Pick up bacteria from plates
Place a drop of water on a slide, then place the loop containing bacteria into the drop. Well suspend bacteria and spread evenly on the slide. Air dry, then fix bacteria on the slides over an alcohol burner.

11 Drop primary stain agent, carbolfuchsin, on bacteria.
Stain for 10 min. Wash excess dye. Add acidic alcohol to decolor for 2 min or until there is no remaining dye. Wash Add secondary dye, methylene blue, to stain for sec.

12 Mycobacterium should be red and other bacteria should be blue.

13 多數之細 菌可藉單染法或革蘭氏染色法而著色，但有少數菌屬尤以分支桿菌屬（Mycobacterium）中之細菌則需藉抗酸性染色法始能觀察。如此屬中之結核桿菌（Mycobacterium tuberculosis）與痲瘋桿菌（Mycobacterium leprae）乃人類之致病性菌，對於此類之鑑定，本染色法極具有診斷價值。 此為傳統染色方式，目前作法是染色為正反應後，在進行DNA及免疫學確定。

14 分支桿菌與其他菌不同，主要系因其細胞壁含有厚層臘質，染料不易滲透。但一經滲透，也不為酸性酒精所脫色。因富此特色，故稱為抗酸性，其他菌則易為酸性酒 精所脫色，乃屬於非抗酸性。

15 初染劑 石炭酸複紅，因多數之分支桿菌對一般染料如甲基藍與結晶紫缺乏感受性，故於複紅中含有石炭酸，如此可使染料溶於細胞壁之脂質成分內， 並使滲透而保留紅色。加熱有助於滲透作用，使石炭酸複紅經細胞壁之脂質而進入胞漿內。齊耳-倪耳生二氏法之改良法則不需加熱，而於染液中加入 潮濕劑，藉以降低細胞壁與染色液間的表面張力，初染後菌體成紅色。

16 脫色劑 酸性酒精（3% HCl+95%乙醇）。脫色前塗抹先使冷卻，俾含臘質之細胞物質硬化。加入酸性酒精時，初因初染劑較脫色劑更易溶於該細胞臘 質，故抗酸性菌對脫色具抗性，因此保留初染劑而成紅色，非抗酸性菌因缺乏細胞臘質，脫色時初染劑易移去而成無色。

17 複染劑 甲烯藍，用以染色脫色之細胞，因只有非抗酸性菌發生脫色，故可吸收複染劑而成藍色，抗酸性菌則仍保留初染之紅色。

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20 Mycobacterium tuberculosis in lung tissue