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Procedure for this week:

Procedure for this week:. Make smears and let air dry Combined and separate bacteria Finish last week’s lab Differential staining introduction Gram stain Acid-fast stain Microscope Review for L ab Practical I. Review from last week: (while your slides air-dry). Review streaks

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Procedure for this week:

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  1. Procedure for this week: Make smears and let air dry Combined and separate bacteria Finish last week’s lab Differential staining introduction Gram stain Acid-fast stain Microscope Review for Lab Practical I

  2. Review from last week:(while your slides air-dry) Review streaks Can you see individual colonies? Can you see organisms off of streak lines? Can you see multiple bacterial colonies on each plate? “Think about it” questions

  3. Differential Staining

  4. Differential Stains • Gram Stains and Acid Fast Stains are differential stains (use more than one color) • The two colors used should be on opposite sides of the color wheel to make visualization easier

  5. 2 Staining protocols this week • Gram Stain (Exercise 7) • Acid-Fast Stain (Exercise 8) Acid fast is RED; non-acid-fast is BLUE Gram (+) is purple; Gram (-) is red

  6. What is a Gram stain? • One of the most important biological staining processes in microbiology • Differential stain • Used to separate many known bacteria (and most medically important ones) into 2 groups • Gram positive • Gram negative • Gram (+) vs. Gram (-) is dependent upon the construction of the bacteria's cell wall • The cell will either retain or lose the first dye

  7. Why these differential stains work • Differences in cell walls • Gram (+): thick peptidoglycan • Gram (-): thin peptidoglycan • Mycobacteria: medium peptidoglycan, pseudo-membrane of mycolic acids and oily lipids (NOT phospholipids) • What are consequences: • Most dyes cannot enter mycobacteria easily, and cannot leave mycobacteria easily • Iodine complexes with multi-layered oligosaccharides

  8. Gram Stain Procedure (p.48) • 1. Make smear, either: • SMALL drop of water + some of colony • Loop of liquid culture • Spread bacteria; make it THIN! • Air dry • 2. heat fix (30 seconds at warm-but-tolerable to hands – 45ºC or 110ºF) • If it is too hot, you will burst your bacteria • If it is too wet, you will boil your bacteria

  9. Gram Stain Procedure • 3. crystal violet addition • flood slide • Let them drown for 1 minute • Rinse slide with water ABOVE smear, let water shower over smear • Crystal violet stains all cells darkly; binds membranes and DNA

  10. Gram Stain Procedure • 4. iodine addition • flood slide • Let them drown for 1 minute • Rinse slide with water ABOVE smear, let water shower over smear • Iodine complexes with oligosaccharide component of peptidoglycan (remember iodine + starch vs. iodine + sugar?)

  11. Gram Stain Procedure • 5. decolorization • Rinse slide with 95% alcohol ABOVE smear, let alcohol shower over smear • Rinse ONLY until most of color has left the slide (a few seconds; do NOT overdo!) • Iodinized peptidoglycan will slow the extraction of crystal iodide from cell, but will NOT stop it (do NOT decolorize forever!) • Rinse slide with water ABOVE smear, let water shower over smear

  12. Gram Stain Procedure • 6. saffranin addition • Flood slide • Let them drown for 1 minute • Rinse slide with water ABOVE smear, let water shower over smear • Saffranin stains all cells darkly; binds nucleic acids and membranes in cells • NOTE that crystal violet will overshadow safranin staining • Bibulous paper • Observe at 100x • Share with labmates • CLEAN YOUR OBJECTIVE LENSES

  13. Acid Fast Procedure (p. 53) • 1. Make smear, either: • SMALL drop of water + some of colony • Loop of liquid culture • Spread bacteria; make it THIN; mix this WELL to minimize clumps • Air dry • 2. Heat fix (30 seconds at warm-but-tolerable to hands – 45ºC or 110ºF) • If it is too hot, you will burst your bacteria • If it is too wet, you will boil your bacteria

  14. Acid Fast Procedure • 3. carbolfuchsin addition • flood slide • Let them drown for 5 minutes • Rinse slide with water ABOVE smear, let water shower over smear • Carbolfuchsin stains all cells darkly; binds membranes, mycolic acids, and DNA X

  15. Acid Fast Procedure • 4. decolorization • Done with acid alcohol (95% alcohol, 1% HCl) • Either: • Rinse ABOVE smear, let alcohol shower over smear, ONLY until most of color has left the slide (may take up to 1 minute) • Flood slide with acid alcohol and let stand for 1 minute X

  16. Acid Fast Procedure • 4. decolorization • Mycolic acids bind carbolfuchsin stronger than they bind other lipids, but acid alcohol will eventually remove carbolfuchsin even from the mycolic acids (do NOT decolorize forever) • Rinse slide with water ABOVE smear, let water shower over smear X

  17. Acid Fast Procedure • 6. methylene blue addition • flood slide • Let them drown for 30 seconds • Rinse slide with water ABOVE smear, let water shower over smear X

  18. Acid Fast Procedure • 6. methylene blue addition • Stains non-acid fast cells blue; binds nucleic acids and membranes in cells • Non-acid-fast cells have ONLY blue • Methylene blue will not penetrate mycolic acid layer under these conditions • Acid-fast cells have ONLY red (why?) X • Bibulous paper • Observe at 100x • Share with labmates • CLEAN YOUR OBJECTIVE LENSES

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