(b) If spore-former grown in liquid broth is to be observed, the broth culture, containing only vegetative cells, is subjected to heat stress, by heating on a water bath for 15 minutes, so that some of the vegetative cells form spores to overcome the heat stress. As the age of spore-forming bacteria on plate or slant cultures increases, they form spores. Then, by gentle rotation of the loop in the water drop, a bacteria suspension is made and the drop is spread till a smear is obtained. A loop is sterilised over flame and a Clostridium loop of bacteria from an aged plate or slant culture (48-72 hours old) of spore-former, such as Clostridium supergenes or Bacillus subtilis is transferred to the water drop. (a) If spore-former grown on agar plate or agar slant is to be observed, a drop of water is placed at the center of the slide. A smear of spore-forming bacteria is made at the center of a slide in two methods as given below. The adhering water is wiped out with bibulous paper and the slide is air-dried.ģ. A slide is cleaned properly under tap water, such that water does not remain as drops on its surface.Ģ. Slide, loop, malachite green, safranin, spore-forming bacteria, hot plate, microscope, immersion oil. When counter-stained with safranin, the colourless vegetative cells take up the stain and appear red unlike the spores, which appear green. Now, the vegetative cells appear colourless. On the other hand, the vegetative bacteria cells take up the primary stain, malachite green, easily, but the stain does not have strong affinity for vegetative cell components, due to which it is washed away, when decolourised under tap water. Thus, finally the spores retain the green colour of the primary stain and appear green. Subsequently, when counter-stained with safranin, the safranin molecules cannot enter into the spores through the covering layers. Thus, the spores retain the green colour. The tap water removes only the excess primary stain present in the surrounding of the spores. ![]() When the spores are treated with the decolourising agent, tap water, they do not undergo decolourisation, as they exclude water. Therefore, penetration of the primary stain is augmented by the application of heat, which drives it through the covering layers into the spore protoplast. The primary stain, malachite green, cannot penetrate into the spores through these layers. The spores are different from the vegetative cells in that they possess thick, relatively impervious layers around them (Figure 2.14). Spore staining is also helpful in identification of the bacteria belonging to the genera Bacillus, Clostridium and Desulfotomaculum. The purpose of spore staining is to differentiate the spores and vegetative cells of a spore-former and to differentiate spore-formers from non-spore-formers. The spore gains its resistance by several mechanisms, which have not yet been clearly explained.Ī bacteria, which cannot produce a spore and therefore, dies in its vegetative form under adverse environmental conditions, is a non-spore-forming bacteria (non-spore-former). The spore has thick, relatively impervious layers, such as spore cortex and spore coat, which protect the cell from any physical damage. The process of spore formation is called ‘sporogenesis’. If adverse conditions worsen, the cell ruptures, releasing the endospore, which now becomes an independent dormant cell called ‘spore’. This spore, formed inside the bacteria cell, is called ‘endospore’ (Figure 2.13). In adverse conditions, a spore is produced within a vegetative cell by dehydration and contraction of its cell contents. However, few species of bacteria, can survive under such adverse conditions by changing themselves to highly resistant, metabolically inactive forms called ‘spores’. On the other hand, most of them die, when the environmental conditions become adverse, such as, severe cold, extreme heat, ageing, lack of nutrients, exposure to radiation and toxic chemicals.
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