All about stains

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Author:
Chesterand19th
ID:
206272
Filename:
All about stains
Updated:
2013-03-10 22:02:24
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Microbiology
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Description:
Smears, negative, simple, gram, acid fast, and endospore stains
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  1. Steps for a negative stain
    • 1) Obtain 1 clean well-washed slide and one dirty #2 slide.
    • 2) Draw a dime-sized circle 3/4 down the side on the bottom.
    • 3) Add 1 drop of Nigrosine to the circled area.
    • 4) Take a flat wooden stick and remove the plaque between the gum and tooth (DO NOT GET ROUGH!) and rub the material on the stick onto the slide in the circled area MIXING it with the Nigrosine.
    • 5) Take the other slide #2 and tip it on its narrow edge.  Place this edge at the beginning of the drop of mixed ink and microbes and lower slide #2 to almost touching slide #1 then DRAG the #2 slide along the #1 slide smearing the black dye down the whole length of the #1 slide.
    • 6) Wash the #2 slide well.
    • 7) AIR DRY the #1 slide and view it under first 10X and then oil immersion.When you are observing the slide DO NOT BE FOOLED BY DRYING DYE CRACKS!  You are looking for faint greyish-white rods and cocci...
  2. What is a negative stain used for?
    to see microbes that are encapsulated. Negative stain provides a dark background to see the white (colorless) capsule.
  3. Why is a capsule beneficial to a microbe?
    • - makes it sticky and prevents it from being swallowed by saliva
    • - makes it undetectable by the immune system, thus protecting the microbe because capsule is made of sugar
  4. How to make a smear
  5. 1) Wash and rinse the slide well or until the water sheets-off the slide; place a small circle about the size of your little fingernail on the underside of each labeled slide at about 75% down the slide with a black sharpee
    • 2) Heat and cool the entire loop, capture a drop of water in the loop
    • 3) apply the drop to the top of the slide at the circle; afterward heat and cool the loop
    • 4) transfer bacteria from an agar surface (DO NOT CHUNK THE AGAR) using a loop.  Obtain the smallest amount of bacteria you can still see in the loop
    • 5) using the loop, mix the bacteria in the water and spread it as widely as possible (it should be JUST barely cloudy when mixed)
    • 6) Air Dry
    • 7) Heat Fix then stain as desired
  6. Steps for a simple stain
    • 1) Obtain a slide with a prepared smear.
    • 2) Lay the slide (Smear side-up) on your staining rack in your sink.
    • 3) Cover the smear with Methylene Blue for 60-120 seconds.
    • 4) Obtain a water spray bottle and aim the water spray ABOVE the smear and wash the excess dye off the smear GENTLY.  DO NOT OVERWASH!
    • 5) Shake the excess water off the slide and blot it dry with bibulous paper.
    • 6) View the slide first under 10X and then oil immersion.
  7. what can you see in a simple stain
    The simple stain is useful in determining the cell size and shape and when different shaped microbes are being examined, it may assist in observing contamination of a pure culture.


    • Extra:
    • As microbes are colorless and somewhat negatively charged, all cationic or positively charged (Basic) dyes may be used for simple staining.  The cationic charged dyes are: Basic Fuchsin, Methylene Blue, and Crystal Violet.
  8. Steps for a gram stain
    • 1) Place the slide with a prepared SMEAR right-side-up on the staining rack in your sink.
    • 2) Cover the circled smear with the PRIMARY STAIN for 1 minute.- crystal violet
    • 3) Obtain a spray water bottle and gently aim the water spray ABOVE the circled smear.
    • 4) SHAKE the excess water off the slide.
    • 5) Return the slide to the staining rack and cover the smear with the MORDANT.  Immediately shake the Mordant off the slide and reapply the Mordant.  Wait for one minute.  Shake the excess mordant off the slide.
    • 6) Return the slide to the staining rack and drop 1-3 drops of ETHANOL on the smear.  QUICKLY and immediately shake it off.  Wash the smear with water as before and shake the excess water off the slide - DO NOT OVER WASH!
    • 7) Return the slide to the staining rack and cover the smear with the COUNTER or SECONDARY STAIN  shake it off the slide and re-apply.  Leave the Counter stain on the smear for 1 minute and then wash GENTLY.  
    • 8) Shake the excess water off the slide and blot the slide dry with BILBOUS PAPER.  DO NOT RUB THE SLIDE!
  9. Is a gram stain a differential stain?
    Yes
  10. Is a negative saturation a differential stain
    No
  11. Is a simple stain a differential stain?
    No
  12. Is an acid fast stain a differential stain?
    yes
  13. Is an endospore stain a differential stain?
    Yes
  14. What does it mean when a microbe stains gram positive?
    Gram positive staining (PURPLE-BLUE) microbes have a VERY thick cell wall made up of either a huge thick layer of peptidoglycan or in a the case of one Genus, a thin peptidoglycan layer with a large thick LIPID layer on top (the Genus MYCOBACTERIUM).  As Crystal Violet dye molecules are very large and the mordant makes them LARGER by cross linking several, the primary stain is IRREVERSIBLY trapped behind the THICK cell wall where the decolorizing ethanol cannot penetrate and remove.  The colorless microbe is stained BLUISH, the secondary pink stain only making the final microbe color PURPLE-BLUE by adding a small amount of pink tint.
  15. What does it mean when a microb stains gram negative?
    Gram negative staining (PINK) microbes have two membranes but a VERY thin cell wall made up of only a few layers of peptidoglycan.  As Crystal Violet dye molecules are very large and the mordant makes them LARGER by cross linking several, the primary stain REVERSIBLY moves behind the THIN cell wall where later the decolorizing ethanol penetrates and removes it.  The colorless microbe is not stained and is now colorless again.  The secondary pink stain is added and NOT removed with a decolorizer making the final microbe color lightPINKISH RED.
  16. What diodes it mean when a microbe stains gram variable on the positive side
  17. Ex:
    the Mycobacteria may take-up the primary stain and loose it at the same time. This microbe is listed as Gram variable on the positive side meaning MOST of the rods will stain purple...
  18. What is the caution when staining the bacillus microbe?
    CAUTION: The Bacillus will often stain as purple but have debris underneath the rods that are staining pink.  As these cells get older (more than 24 hrs old) they fall apart and the pieces stain pink... the stain should be read as Gram positive
  19. What are the steps for an acid fast stain?
    1.     Make a typical smear.

    2.     After heat-fixing, cover the circled smear with Carbolfuchsin stain.  DO NOT PUT TOO MUCH STAIN.

    3.     Hold the slide over the bunsen burner until the the slide gets HOT AND about 20% of the primary stain has evaporated (NOT ALL OF IT!!!!!).  Constantly move the slide in and out of the flame or up and down. Put paper towel under the Bunsen burner to catch any "spills." SHAKE off the excess PRIMARY STAIN after heating... then...

    4.   Put 1/2 dropper full of ACID ALCOHOL on the smear and immediately wash it off with water...DO NOT OVERWASH!

    5.     Counter stain with METHYLENE BLUE for 3-4 min. or so.  Wash with water... Blot dry, add immersion oil and focus under 10X

    6.   View under oil immersion – 100X, draw.
  20. Why was the acid fast stain developed?
    The Acid Fast Stain was developed for the GENUS MYCOBACTERIUM which contain species that cause Tuberculosis, Leprosy, and other fatal illnesses.   We use one of two non-pathogenic Mycobacterium in our classes for demonstration.   Mycobacterium are notoriously hard to kill and hard to stain; they also grow very very slowly.  All of these characteristics of the microbe are due to the veryTHICK LIPID LAYER in the cell wall.  As you might expect, the Mycobacterium stain Gram-positive because of the THICK cell wall.  REMEMBER... the peptidogycan layer in Mycobacteria is THIN but it stains Gram variable on the positive side (almost all Gm+).
  21. What makes a culture acid fast?
    CAUTION: 1- ONE HOT PINK ROD makes the stain Acid Fast (otherwise known as Acid Fast Positive).  This is because Mycobacteria are hard to stain... you will see clumps of rods... and only a few are fuchsia or hot pink.
  22. How do you read endospores in an acid fast stain?
    CAUTION: sometimes the ENDOSPORES will take-up the primary stain and appear like HOT PINK ovals.. this is still an acid fast negative or non-acid fast stain because we are staining CELLS.... endospores are NOT CELLS and thus do not count.
  23. What does it mean when aculture is acid fast?
    Principle:  Some bacteria contain a waxy lipid, mycolic acid, in there cell wall.  This lipid makes the cells more durable and is commonly associated with pathogens.  Acid fast cell walls are so durable that the stain (carbol fuschin) must be driven into the cells with heat.  The cells are then decolorized with acid-alcohol, all other cells will decolorize with this strong solvent, but acid fast bacteria will not.
  24. What does it mean when a culture is non acid fast?
    Acid fast cell walls are so durable that the stain (carbol fuschin) must be driven into the cells with heat.  The cells are then decolorized with acid-alcohol, all other cells will decolorize with this strong solvent, but acid fast bacteria will not.  Other cells are then counterstained with methylene blue.
  25. Steps for an endospore stain
    1.     Make a typical smear... .but put the circle about 3/4th of the way down the slide to allow you to hold the slide while heating the primary stain

    2.     After heat-fixing, cover the smear with malachite green.  DO NOT PUT TOO MUCH STAIN.

    3.     Pass the slide through the Bunsen burner 4-5X until the slide is VERY WARM (the "edge" of the bubble of dye liquid begins to evaporate)!!  Constantly move the slide in and out of the flame or up and down.  Put a piece of paper towel under the Bunsen burner to catch "spills."

    4.     Rinse the malachite green off the slide with water until the run-off is clear, aim the water above the smear DO NOT OVERWASH!5.     Counter stain with Safranin for 3-4 min. or so.  Wash with water (DO NOT OVERWASH), Blot dry, add immersion oil and focus under 10X
  26. Why was the endospore stain developed?
    The ENDOSPORE STAIN was developed for the rod-shaped GENERA BACILLUS (aerobic) & CLOSTRIDIUM (anaerobic) as well as a for few other rare Genera with endospores.
  27. What are endospores?
    ENOSPORES are extreme structures!  They are almost impossible to destroy!  They resist: heat, cold, freezing, drying, radiation, and chemicals.  They are NOT reproductive structures (as a similar word "spore" is in plants and fungi) but are SURVIVAL structures in bacteria.  Each vegetative cell may make one spore which under favorable conditions will germinate into one cell...  Most endospore producing rods stain Gram-positive because of their THICK cell wall.  Endospores are very very difficult to stain and thus most endospore stains require HEAT or STEAM.
  28. When is a endospore more visible in an endospore stain?
    CAUTION: ENDOSPORES are present at all times in a culture containing "spore" producing microbes, but MORE endospores are visible when conditions are unfavorable to the microbe!
  29. What should you do if you see rods in an endospore stain?
    CAUTION: Endospores are never "rods" so if you see pink and green RODS it is negative for endospores... Endospores are round or "egg-shaped" or may be seen as "EMPTY" holes in a pink rod...
  30. what happens if you don't see any pink rods in an endospore stain?
    CAUTION: An entire culture may revert to its ENDOSPORE stage and thus appear as hundreds of green oval-shaped endospores without the pink vegetative rod-shaped cells that produced them!

    still positive!
  31. Size of an endospore compared to the vegetative cells?
    CAUTION: Endospores are ALWAYS SMALLER THAN THE VEGETATIVE CELL THAT PRODUCED THEM!
  32. What does it mean when a culture stains endospore positive?
    The ENDOSPORE STAIN utilizes MALACHITE GREEN (primary stain) which is a green stain that has an affinity for the Calcium ions in the endospore coat.  The counterstain is SAFRANIN.  SP+ microbes vegetative cells stain as pink colored rods while internal endospore or released smaller round or egg-shape endosproe stains GREEN.  Sp- microbes vegetative cells all stain PINK.
  33. What does it mean when an endospore stain reads negative?
    No spores are present
  34. What happens if your entire endospore stain is green?
    Another common error is having EVERYTHING on the slide stain GREEN.  You allowed the slide to go dry while boiling or boiled too fast and too hot!THERE IS NO DECOLORIZER USED! NO ALCOHOLS!Don't over-wash or under-wash!
  35. Why is scanning the entire slide important in an endospore stain?
    A microbe is considered SPORE Positive when ANY smaller internal or released egg-shaped or round SPORES stain GREEN. Endospores are very difficult to stain - you must SCAN the whole slide to decide.

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