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4 Cards in this Set

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Gram Stain

Primary Stain: "Crystal Violet", binds to cell-all cells stain purple. Stain for 1min


Mordant: "Iodine", forms insoluble complex w/purple stain. Apply for 1min


Decolorizer: "95% Ethyl Alcohol", destains gram negative bacteria/gram positive remain purple. Apply for 15-20sec


Counterstain: "Safranin", destained gram neg bacteria stain pink/red. Counterstain for 1min, then blot with bibulous paper.


Organism used E. Coli and S. Aureus

4 steps

Negative Stain

6 slides


Organism used, M. Luteus and B. Cereus


1. Loopful of Nigrosin close to one end of clean slide


2. Place loopful of bacteria to drop of Nigrosin, avoid label and DONT HEAT FIX, mix well.


3. With edge of second slide held at 30-degree angle and placed in front of the bacterial suspension, push the mixture to form a thin smear.


4. Air dry, don't heat fix, Do Not BLOT!!


5. Examine slides under oil, measure diameter of M.Luteus & length vs. width of B. Cereus w/ocular micrometer (measure=5 of each bacteria, average, and then round-off.)


Make only one slide at a time, the loopful.of Nigrosin dries out.

5 steps with 2 slides per

Spore stain

Organism used; B. Cereus-Gram + rod


B. Subtilis-Gram + rod


C. Sporogenes-Gram + rod


1. Make a heat fixed smear


2. Place a piece of paper towel on the top of bacterial smear


3. Add Malachite Green and heat over water until steam(5mins) add more Malachite Green do not let stain dry out.


4. Cool slide, after cool wash with tap water.


5. Counterstain w/ Gram's Safranin stain for 2mins


6. Wash with water and blot dry w/ bibulous paper


Primary stain: Malachite Green


Decolorizer: Distilled Water


Counterstain: Gram's Safranin

6 steps

Acid Fast Stain

Organism used; M. Gordonae & S. Aureus


1. Prepare thin bacterial smear. Air dry or use slide warmer.


2. Heat Fix Smear!!! Don't forget.


3. Flood smear w/Carbol Fuchsin for 5 mins


4. Wash well w/water, shake off excess, Do Not spray directly on organism.


5. Decolorize w/Acidified alcohol for 3-5sec.


6. Wash w/water shake off excess.


7. Counterstain w/ Methylene Blue for 5mins


8. Wash w/distilled water


9. Air dry. Examine using oil immersion.

Remember 3 types of drops needed.