SMEAR THICKNESS!!!
THE ENDOSPORE or "spore" STAIN:
Atlas page 32
NOTE: "CAUTIONS" on interpretation below!!!!
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  | Pink cells with empty holes are Endospore positive! PHOTOS of ENDOSPORE positive cultures... the endospores are smaller than the cells that made them. The vegetative cells stain pink or red and Endospores stain GREEN. |
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.
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.
INTERPRETATION CAUTIONS:
CAUTION: ON AN ACID FAST STAIN THE ENDOSPORES may take-up the Acid Fast Primary Stain and look fuchsia, but since they are NOT cells they are NOT Acid Fast Positive!
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!
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!
CAUTION: Endospores are ALWAYS SMALLER THAN THE VEGETATIVE CELL THAT PRODUCED THEM!
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...
SMEAR from agar begins all stains - the STEPS:
ALL STAINS START WITH THE PROPER PREPARATION OF A SMEAR (from agar usually):FIRST PREPARE A GOOD SMEAR FROM AGAR:
1. Disinfect your table-top and obtain your incomplete Universal Precautions Lab Clothing. Clean a slide WELL, dry the cleaned slide. Using your Sharpee, place a dime-sized circle about 3/4th of the way down on the BOTTOM SIDE of the slide...
2. Heat your loop to red hot and cool it...
3. "Capture" a drop of water on your loop...
4. Spread the water on top of the circled area of the slide..
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5. Heat your loop to red hot and cool it...
6. Obtain a culture on a test tube agar slant (or from a colony on an agar plate - for other preps see "Preparation of a Smear from a Broth")...
7. Flame the test tube lid gently, remove the lid with your "pinky-finger" of the hand holding your loop and hold the lid in your hand (NEVER lay a top or loop on a table top!). Flame the LIP of the test tube gently. Insert your loop into the test tube and go PAST the slant where the microbe is growing on the agar as a "film." Lower the loop and gently pull it toward you as it glides gently across the SURFACE of the slant picking-up a small amount of gelatinous microbe film inside the loop. DO NOT GET THE AGAR GEL ON YOUR LOOP!
1. Disinfect your table-top and obtain your incomplete Universal Precautions Lab Clothing. Clean a slide WELL, dry the cleaned slide. Using your Sharpee, place a dime-sized circle about 3/4th of the way down on the BOTTOM SIDE of the slide...
2. Heat your loop to red hot and cool it...
3. "Capture" a drop of water on your loop...
4. Spread the water on top of the circled area of the slide..
.
5. Heat your loop to red hot and cool it...
6. Obtain a culture on a test tube agar slant (or from a colony on an agar plate - for other preps see "Preparation of a Smear from a Broth")...
7. Flame the test tube lid gently, remove the lid with your "pinky-finger" of the hand holding your loop and hold the lid in your hand (NEVER lay a top or loop on a table top!). Flame the LIP of the test tube gently. Insert your loop into the test tube and go PAST the slant where the microbe is growing on the agar as a "film." Lower the loop and gently pull it toward you as it glides gently across the SURFACE of the slant picking-up a small amount of gelatinous microbe film inside the loop. DO NOT GET THE AGAR GEL ON YOUR LOOP!
CURRENT (ALTERNATE) PROCEDURE FOR THE ENDOSPORE STAIN:
PROCEDURE:
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 - make a "bubble" of stain - DO NOT PUT TOO MUCH or too little 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
Any stain may be an Endospore Stain
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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.
COMMON ERRORS IN ENDOSPORE STAINING:
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.
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!
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WE USE THE ALTERNATE PROCEDURE FOR THE ENDOSPORE STAIN!
View under oil immersion – 100X, draw.
| Endospore positive Bacillus.. note red vegetative cell with empty "holes" as well as the green stained endospores within the rods... | PHOTOS of several ENDOPSORE STAINS - ours most looks like rods with pink vegetative cells and light green smaller endospore (or empty holes) so IGNORE GREEN! |