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Gram staining Techniques

Gram staining Techniques. Some history. Bacteria are translucent Staining make them visible under microscope 1884 Hans Christian Gram  stained cells and found that some lost their color when excess stain was washed off Differential stain  distinction between 2 types of bacteria.

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Gram staining Techniques

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  1. Gram staining Techniques

  2. Some history • Bacteria are translucent • Staining make them visible under microscope • 1884 Hans Christian Gram  stained cells and found that some lost their color when excess stain was washed off • Differential stain  distinction between 2 types of bacteria http://www2.bvs.org.ve/img/fbpe/rsvm/v23n2/Image140.jpg

  3. Bacteria cell walls • Peptidoglycan: network of sugars cross-linked by short peptides • Forms the rigid part of the cell wall • Protects the bacteria against mechanical damage • Part that picks up the stain in the gram procedure www-micro.msb.le.ac.uk/ video/graphics/wall.gif

  4. Gram - bacteria • Small peptidoglycan • Are stained with crystal violet but decolorized with alcohol after which they pick up the red stain • LPS on outer membrane  toxic for host http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/prostruct/toll/u1fig10b.html

  5. Gram + bacteria • Large, highly cross-linked petidoglycan • No outer membrane http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/prostruct/u1fig9b.html

  6. gsbs.utmb.edu/microbook/ images/fig2_6.jpg

  7. Bacteria shape • Long rod: bacillus • Round shaped: cocci • Spiral: Spirochete

  8. Bacteria shapes http://ibri.org/RRs/RR051/51bacterialshapes-Merc.gif

  9. Some bacteria shapes spirochete bacillus coccobacillus cocci http://www.spcollege.edu/hec/vt/VTDE/ATE2639LGS/images/image11.jpg http://textbookofbacteriology.net/B.anthracis.Gram.CDC.jpeg http://www.furetti.com/images/spirochete%20leptospirosi%201.jpg

  10. Gram stain • Is the most commonly used technique to stain bacteria • Almost always first step into identification • Tell what other tests to perform • Gram – bacteria stain red-pink • Gram + bacteria stain blue-purple

  11. Gram Positive Bacteria (blue/purple) Gram Negative Bacteria (Red/pink)

  12. Gram stain recipe • Smear • Heat fix • Crystal violet (30 sec)  wash • Iodine (1 minute)  wash • Ethanol (varies ~15 sec)  wash • Safranin (30 seconds)  wash • Blot dry

  13. How to prepare a smear • Label your slide with a pencil • Use a clean slide: manipulate by the edges • Using a sterilized loop spread of drop of bacterial culture on the slide • If solid culture  put a drop of water on the slide  add a little bit of bacteria (using loop) • A thin film is better • Dry faster, distribution of dye and decolorizer more even • No coverslip on! let dry (until most water evaporated)

  14. Fixation • Pass the slide through the flame of the bunsen burner • Moving in a circular motion (to avoid localized overheating) • The slide should not be too hot to touch, slightly warm but no more! • If too hot  overfixed  burn bacteria everything will be black • If underfixed  bacteria do not stick to slide and will be washed away during the staining procedure

  15. Why bacteria need to be fixed • Denature bacterial enzyme  prevent them from digesting the cell (autolysis) • Make the bacteria stick to the slide so they are not washed away during staining.

  16. 1st stain • Crystal violet • Colorize all cells • Flood the slide with crystal violet • ***make sure that the bacteria are on top • 30 seconds • Rinse gently with running tap (or distilled) water • Do not squirt water directly onto the smear • Shake off the excess water

  17. Mordant • Iodine • Make the dye stick to the cell wall • Crystallize the dye in the peptidoglycan • Flood the slide for 1 minute • Wash

  18. Decolorization • Ethanol 15-30 seconds, until dye doesn’t run out anymore • Critical step***** Washing after is very important (stop alcohol action) • Cell that have thin peptidoglycan decolorize • Long story: • Dissolve the lipid layer from the gram negative bacteria • Enhance the leaching from the primary stain • In gram + bacteria: alcohol dehydrate the thicker cell wall • Prevent diffusion of the violet iodine complex

  19. Ethanol Sink

  20. Counter stain • Safranin •  color pink • Flood the slide for 30 seconds • Rinse gently with water and shake off the excess water from surface

  21. Blotting • Slides can be air dried or blotted • Blotting  put between 2 sheets of absorbent paper (we will use bibulous paper). • DO NOT RUB THE SMEAR (bacteria will come off)…just blot between papers.

  22. Oil immersion • Light is refracted when goes trough slide (change in media from glass to air) • Oil has same refractive index than glass  light ray goes with no refraction • Use only one drop of oil • Only use the 40X objective (greatest power objective) with oil immersion. • Clean the objective with paper lens after use

  23. Oil immersion http://www.bmb.psu.edu/courses/micro107/microscopy/oil-lens.jpg

  24. Gram Staining Video • http://www.youtube.com/watch?v=YvZHHNZ8cdo • http://www.bio.upenn.edu/computing/media/Instructional.Stain.Gram.php

  25. Objectives for Tomorrow’s Lab • You have 2 bacteria • S. Urea • P. fluorescens For each bacteria: • Stain each bacteria • Observe and identify the bacteria shape and gram results. • You are doing this lab in team but make sure that each student perform at least 1 gram stain

  26. Resources • www.microvet.arizona.edu/Courses/MIC205/lab/Lab_3_Gram_stain_spring_07.ppt • www.google.com/images/

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