Biology and Biotechnology department Differential stain Biology and Biotechnology department

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

Gram Stain : Differentiate bacteria into 2 groups: Gram positive bacteria Gram negative bacteria

differences between gram positive and gram negative bacteria Gram positive bacteria Thin cell wall ( one layer of peptidoglycan ) Thick cell wall (many layers of peptidoglycan) Absent Teichoic acids : Lipoteichoic acids link to the plasma membrane Wallteichoic acids link to the peptidoglycan ROLE: Provide rigidity to the cell wall.

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

Gram Stain components Like most differential staining procedures has : Primary stain , mordant and / or selective treatment (decolorizer), and counter stain . 1. Primary stain : colors the target cells or cell components. Here the primary stain is crystal violet & the target cells are the 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 )

95% ethanol rinse is used here to remove excess crystal violet 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 Safranin is used.

Procedures of Gram Stain 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 Safranin (30 sec) Dry mirsope

result

Gram positive bacteria : Bacillus , streptococcus Notes : Examples : Gram positive bacteria : Bacillus , streptococcus Gram negative bacteria : E.coli 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

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+ G- low :G- G+ Time of fixation (mordant ) : low : no sample remain on the slide after wash

B. Acid-fast Stain Differentiate bacteria into 2 groups: Acid-fast bacteria: retain of the basic stain in the presence of Acid-alchole 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: lose the basic stain when rinsed with Acid-alchole and usually counterstained to see them Don't have waxy lipid

Acid-fast bacteria are pathogenic : Mycobacterium tuberculosis tuberculosis Mycobacterium leprae leprosy 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 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 :