Endospore Stain PDF

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Microbiology 2420 Lab Report on Endospore Staining...

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Abstract Endospore Staining is a technique used in bacteriology to identify the presence of endospores in a bacterial sample, which can be useful for classifying bacteria. Within bacteria, endospores are protective structures used to survive extreme conditions, but this protective nature makes them difficult to stain using normal techniques such as simple staining and Gram staining. Specially, the Schaeffer-Fulton Stain method is used for this experiment in which heat is used to drive primary stain: Malachite green into the spores’ coat and counter stain: Safranin. The vegetative cell containing endospores which appear in pink or reddish color, on other hand the spores appear in green color.

(Keyword: Vegetative cell, Spores and Endospore staining)

Introduction An endospore is a dormant, tough, and non-reproductive structure produced by certain bacteria from the Firmicute phylum. The name "endospore" is suggestive of a spore or seed-like form (endo means within), but it is not a true spore (i.e. not an offspring). Endospore formation is usually triggered by a lack of nutrients, and usually occurs in Gram-positive bacteria. In endospore formation, the bacterium divides within its cell wall. Most types of bacteria cannot change to the endospore form. Examples of bacteria that can form endospores include Bacillus and Clostridium. Endospores can survive without nutrients. They are resistant to ultraviolet radiation, desiccation, high temperature, extreme freezing and chemical disinfectants. Common anti-bacterial agents that work by destroying vegetative cell walls do not affect endospores. Basically, endospore stain is a differential stain. As such, it allows for the differentiation of structures and as a result the characterization of a cellular based on its physical and chemical nature. In this example, endospore stain as a differential staining approach is largely used for the purposes of distinguishing among vegetative cells and endospores. The main purpose of

Endospore staining is differential stain used to detect the presence and location of spores in bacterial cells. Endospores are formed by a few genera of bacteria, such as Bacillus. The Structure of endospore is very complex since they possess multilayer coverings. The outer layer of the spore is called exosporium which is relatively thin and delicate, beneath the exosporium is a spore coat composed of several layers of proteins. Spore coat is comparatively thick because of thickness of spore coat endospore resistant extreme heat, radiation and chemicals. The inner spore coat is cortex. Cortex is thick layer in endospore which is composed of peptidoglycan. The inner most layer of spore is called Spore cell wall or Core cell wall. Spore of the cell wall cover the cores of the endospore. Bacillus subtilis is Gram positive and is a spore forming, motile. Structurally, Bacillus subtilis is rod-shaped and facultative aerobe bacterium ( Choi, 2015). It is mostly found in soil and vegetation with an optimal growth temperature from 25-35 degrees Celsius. B. subtilis has the ability to produce and secrete antibiotics.

Methods and Materials The smear of Bacillus subtilis was obtained which was made week 4. First of all, prepare smears of organisms to be tested for presence of endospores on a clean microscope slide and air dry it. Heat fix the smear. Place a small piece of blotting paper (absorbent paper) over the smear and place the slide (smear side up) by placing the boiling water. Continually flooded malachite green on slide so that is does not dry or evaporate for 5 to 8 minutes. After 5 minutes carefully remove the slide from the rack using a clothespin. Remove the blotting paper and drain the slide then gently rinse off MG with DI water from squeeze bottle (to wash the malachite green from both sides of the microscope slide). Stain the smear with safranin for 1 minutes over staining

tray. Again drain and wash with water. After blot the slide carefully, the slides were ready to observe under the microscope.

Result From the result that has been obtained in the laboratory, when visualized under microscope the cells have three characteristics, the vegetative cells should appear pink/red, the vegetative cells that contain endospores should stain pink while the spores should be seen as green ellipses within the cells. Mature, free endospores should not be associated with the vegetative bacteria and should be seen as green ellipses. When we visualized under the microscope at 1000x total magnification with oil immersion, we are able to vegetative cell and endospores clearly. The vegetative cells appeared in pink/red color and the spores appeared in green color.

Endospore Staining results of Bacillus Subtilis taken at 40x total magnification.

Endospore Staining results of Bacillus Subtilis taken at 1000x total magnification under oil immersion. Discussion From observations obtained in the staining bacteria Bacillus subtilis spores, it can be concluded that all the coloring treatment effect on bacterial spores staining results. As we know that the warming time greatly affect the dye to penetrate the walls of bacterial spores this can be seen in the results of staining. Heating time is 5 to 8 minutes’ bacterial spores stained. This is caused because the heating time is longer make the pores of the spore wall is open so as to facilitate the dye to get into the bacterial spores. And We were satisfied with the results obtained from this technique. It was appeared to be the pink or reddish color for vegetative cells and green color for endospore. And the structure was matched. However, we had to face little difficulties

while under the microscope with oil immersion. The organism was not visualized clearly. And our instructor notified us that we use too much oil....