Endospore Stain Lab

Microbiology Lab Notebook Report Endospore Stain - Exercise 3-8 March 3, 2020 Introduction Some bacterial species are able to differentiate into dormant cells called endospores when environmental conditions, such as nutrient depletion or high temperatures, are unsuitable for growth. Endospores are highly resistant to heat and chemicals, which allows them to survive in this state for long periods of time. The total absence of ATP within endospores is an indication of how dormant they are! Purpose The purpose of this lab is to visualize and study endospores prior to and after the counterstain. This allows us to see if the lab and staining ages the endospores. The keratin contained within the endospore coating may impact the absorption of the stain itself. Materials • • • • • • • • • • • • • • Microscope Clean glass microscope slides Malachite green stain Safranin stain Squirt bottle with water Bibulous paper Staining tray Staining screen Slide holder Heating apparatus Immersion oil Lens paper Non-sterile petri dish for transporting slides Recommended organisms 1 Procedure 1. Begin with up to three heat-fixed emulsions on one slide. Wearing gloves and chemical eye protection, cover the smear with a strip of bibulous paper cut slightly smaller than the slide. If you need to transport the slide to another part of the lab for staining, put it in a covered Petri dish. 2. Set the slide on a steaming apparatus and saturate the bibulous paper with malachite green stain. Heat it to steaming for 5 to 7 minutes. Be sure to keep the paper moist with stain, but don't have it so wet that stain runs off the slide. Perform this step with adequate ventilation, preferably in a fume hood. 3. Grasp the slide with a slide holder and properly dispose of the bibulous paper with forceps (to prevent stain from getting on your gloves). Then, hold the slide on an angle over a stain tray and gently rinse both sides with distilled water until the runoff is clear. Then, if not already there, return to your lab station carrying your slide in a Petri dish. 4. Place the slide on the staining rack and counterstain with safranin for 1 minute. Be sure excess stain falls into the staining tray. 5. Grasp the slide with a slide holder and hold it on an angle. Gently rinse the slide with distilled water into the staining tray. 6. Gently blot dry in a tablet of bibulous paper or paper towels. (Alternatively, a page from the tablet can be removed and used for blotting.) Do not rub. When dry, observe under oil immersion. 2 Results Bacillus megaterium Clostridium tetani Clostridium butyricum Observations and Interpretations Organism Cellular Morphology and Arrangement Bacillus megaterium Bacilli shaped, arranged in chains Present -centrally positioned -elliptical -do not cause mother cell to distend/swell at all Clostridium tetani Bacilli shaped, with no particular arrangement Present -terminally positioned -cause ends of the cells to swell -elliptical Bacilli shaped, arranged in single cell structures Present -subterminally positioned -elliptical -spores distend the mother cell Clostridium butyricum Spores (present or absent) Spore Shape, Position, and Dimension Conclusion It is necessary to use steam because it allows the stain to absorb into the keratin of the endospore. I saw more alone endospores than I did endospores attached to vegetative cells.This means that the environment prior was already unfavorable for the cells. 3 Follow-Up Questions 1. Why does this exercise call for an older (48-hour or 5-day) culture of Bacillus? -Young cultures of spore-forming microbes may not demonstrate any endospores because the vegetative cells may not have been subjected to sufficient stress to stimulate sporulation. 2. Consider the possible results of an endospore stain. a. What does a positive result for the endospore stain indicate about the organism? -If you get a positive result for the endospore stain, it indicates (barring contamination) that the organism produces spores. b. What does a negative result for the endospore stain indicate about the organism. -A negative result for the endospore stain might mean the organism cannot produce spores, or CAN and just ISN’T. 3. Why is it not necessary to include a negative control for this stain procedure? -Endospore stain is a differential staining procedure that allows to see both spores and vegetative cells, thus including separate -ve control consisting of only vegetative cells is not required. 4. Endospores do not stain easily. Perhaps you have seen them as unstained white objects inside Bacillus species in other staining procedures. If they are visible as unstained objects in other stains, of what use is the endospore stain? -When the structure is an unstained white object, it might be a spore, or it might be a storage granule or other cellular inclusion of some sort. The spore stain technique provides good evidence that the structure stained is, in fact, a spore. 4