| Technique | SPORE STAINS ,(/,)/,( |
| Method | See Cold Method See Hot Method See Muir’s Procedure |
| Positive control | Bacillus megaterium (ATCC 12872) Bacillus macerans (ATCC 8244) Clostridium butyricum (ATCC 19398) Bacillus circulans (ATCC 4513) |
| Negative control | Escherichia coli (ATCC 11229) |
| Technique | SPORE STAIN "Cold" Method |
| Principle | |
| Cautions | The time required for staining with
malachite green may vary from 10- 20 minutes This time may be shortened to 1 minute by Steaming the slide |
| Method | Fix the smear by passing the slide
through a flame. Stain for 10 Minutes with malachite green without heat Rinse with tap water for about 10 seconds. Counterstain with aqueous safranin (0.25-0.5%) for 15 seconds. Rinse with water and drain or blot dry. |
| Results | The spores will be green And the rest of the cell will be red. |
| Reagents | Malachite Green Solution Safranin Solution |
| Reference | Schaeffer & Fulton 1933 Science 77:1941 |
| Technique | SPORE STAIN "Hot" Method |
| Principle | |
| Cautions | Decolorization of the vegetative cells can best be controlled by examining the wet film under low power magnification |
| Method | Flood the slide with strong
carbol-Fuchsin and steam. After 5 minutes wash the slide well with water. Decolorize with ethanol until all traces of red have been removed. Wipe the BOTTOM of the slide dry to remove excess stain. Wash thoroughly in water Counterstain with Loeffler's methylene blue for 1-2 minutes. Wash and drain or blot to dry. |
| Results | The spores stain red while the bacterial bodies stain blue |
| Reagents | Carbol Fuchsin Solution Loeffler’s Methylene Blue Solution |
| Reference |
| Technique | SPORE STAIN Muir’s Procedure |
| Principle | |
| Cautions | |
| Method | 1:Flood with strong carbol-fuchsin
and heat gently for 1 minute. 2:Rinse rapidly with ethanol, 3:Wash well with water. 4:Flood with Muir's mordant for 30 seconds. 5: Wash off with water, 6: wash with ethanol for 30 seconds or until the film is pink. 7: Wash well with water. 8: Counterstain with Loeffler's methylene blue for 30 seconds 9: Wash and drain or blot to dry. |
| Results | Bacteria stain red while spores stain green |
| Reagents | Carbol Fuchsin Solution Muir’s Mordant Loeffler’s Methylene Blue Solution |
| Reference |