DIFFERENTIAL STAIN Biology and Biotechnology department Differential stain

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DIFFERENTIAL STAIN Biology and Biotechnology department

DIFFERENTIAL STAIN Biology and Biotechnology department

Ø Differential stain group. separate bacteria into Differential stain Gram Stain Acid-fast Stain

Ø Differential stain group. separate bacteria into Differential stain Gram Stain Acid-fast Stain

A. GRAM STAIN : Ø Differentiate bacteria into 2 groups: § Gram positive bacteria

A. GRAM STAIN : Ø Differentiate bacteria into 2 groups: § Gram positive bacteria Gram negative bacteria §

DIFFERENCES BETWEEN GRAM POSITIVE AND GRAM NEGATIVE BACTERIA Gram positive bacteria Gram negative bacteria

DIFFERENCES BETWEEN GRAM POSITIVE AND GRAM NEGATIVE BACTERIA Gram positive bacteria Gram negative bacteria Thick cell wall (many layers of peptidoglycan) Thin cell wall ( one layer of peptidoglycan ) Teichoic acids : • Lipoteichoic acids the plasma membrane • Wallteichoic acids the peptidoglycan Absent link to v ROLE: Provide rigidity to the cell wall.

Gram positive bacteria Gram negative bacteria No outer membrane (a membranous structure surround the

Gram positive bacteria Gram negative bacteria No outer membrane (a membranous structure surround the peptidoglycan , part of the capsule). v ROLE: Resistance to the antibiotic No periplasmic space (between outer membrane and the plasma membrane Mycolic acid (waxy-lipid) Found only in acid fast cells No Mycolic acid

GRAM STAIN COMPONENTS Like most differential staining procedures has : Primary stain , mordant

GRAM STAIN COMPONENTS Like most differential staining procedures has : Primary stain , mordant and / or selective treatment (decolorizer), and counter stain. Ø 1. Primary stain : colors the target cells or cell components. Here the primary stain is crystal violet & the target cells are thick-walled bacterial cells. 2. Mordant : react with the primary stain and the target cells so that the target cells retain the stain (fixation of stain). Here the mordant is Gram's iodine (solution of iodine and potassium iodide )

3. selective treatment (decolorizing agent ) : Cause the target cells to retain the

3. selective treatment (decolorizing agent ) : Cause the target cells to retain the primary stain while removing it from the non-target cells 95% ethanol rinse is used here to remove excess crystal violet 4. Counter stain is which color every things wasn't colored by the primary stain Ø Safranin is used.

PROCEDURES OF GRAM STAIN Ø Ø Ø Smear of bacteria. Stain by crystal violet

PROCEDURES OF GRAM STAIN Ø Ø Ø Smear of bacteria. Stain by crystal violet (30 -60 sec) Washing by D. W Mordant iodine (60 sec) Washing decolorizing agent 95% ethanol drop by drop Washing Safranin (30 sec) Washing Dry mirsope

RESULT

RESULT

 Notes : Ethanol In (G + ve) Dehydrates of peptidoglycan So CV-I crystals

Notes : Ethanol In (G + ve) Dehydrates of peptidoglycan So CV-I crystals donot leave In (G-ve) Dissolve outer membrane & holes in peptidoglycan So CV-I crystals wash out Examples : Gram positive bacteria : Bacillus , streptococcus Gram negative bacteria : E. coli

Ø Ø Ø Never used old culture Never used sample for patient take antibiotic

Ø Ø Ø Never used old culture Never used sample for patient take antibiotic (antibiotic distraction of cell wall) No cell wall ex: Myoplasma Mycopatrium (g +ve) but have waxy lipid so cannot stain with gram but stain with acid-fast stain. Time of decolonization : over : G+ Glow : GG+ Time of fixation (mordant ) : over : G+ Glow : no sample remain on the slide after wash

B. ACID-FAST STAIN Differentiate bacteria into 2 groups: Acid-fast bacteria: Ø retain of the

B. ACID-FAST STAIN Differentiate bacteria into 2 groups: Acid-fast bacteria: Ø retain of the basic stain in the presence of Acid-alchole Ø The cell wall of these bacteria have high concentration of waxy lipid (mycolic acid), macking simple stain & gram stains useless (all acid-fast bacteria are gram +) (Difficult to stain & if stained difficult to remove the bacteria: Ø lose the basic stain when rinsed with Acid-alchole and usually counterstained to see them Ø Don't have waxy lipid Ø

Acid-fast bacteria are pathogenic : Mycobacterium tuberculosis Mycobacterium leprae leprosy Ø Acid-fast Stain components:

Acid-fast bacteria are pathogenic : Mycobacterium tuberculosis Mycobacterium leprae leprosy Ø Acid-fast Stain components: 1. Primary stain is carbol fucsin 2. Decolorizing agent is Acid-alchole 3. Counter stain is methylen blue

PROCEDURES OF ACID-FAST STAIN : Ø Ø Ø Ø Ø Smear of bacteria Carbol

PROCEDURES OF ACID-FAST STAIN : Ø Ø Ø Ø Ø Smear of bacteria Carbol fucsin + tergitol (wetting agent) cover bacteria 5 mines if tergitol is not available Carbol fucsin cover bacteria place slide on pre-warmed hot plate 5 mines These facility the penetration of waxy lipid Acid-alchole drop by drop Washing by D. W M. B 1 -2 mines D. W Dry mirsope

RESULT :

RESULT :