Endospore Test Lab Report

The catalase test is used to differentiate staphylococci (catalase-positive) from streptococci (catalase-negative). The enzyme, catalase, protects the bacteria from the toxic by-products of oxygen metabolism. This enzyme is produced by bacteria that respires using oxygen. The catalase-positive bacteria include strict aerobes. Catalase-negative bacteria may be anaerobes, or they may be facultative anaerobes do not respire using oxygen as a terminal electron acceptor. The test reaction is very fast and obvious bubbles will be seen. 30% Hydrogen peroxide will be drop on a glass slide. The inoculum of 24 hours fresh culture will be picked by using sterile loop and placed on the slide that contain H²O² drop. Bubble formation on the slide will show
The primary dye malachite green is a weakly binding dye to the spore wall and cell wall. The dye will be locked in the spore wall, which has peptidoglycan deeper in the walls. The keratin forming the outer portion of the endospore wall resists dye. The heating of the bacteria will make the spore wall more permeable to the malachite green, and it then attaches to the peptidoglycan. Thus, we can detect the bacteria that have the endospore based on this test. On a clean glass slide a drop of distilled water will be added to the slide. A smear will be prepared on this droplet by using fresh culture of bacteria and then will be air dried and heat fixed. The smear will be flooded with malachite green (0.5% aqueous solution). The slide will be heated so that malachite green will be steamed for 5 min but will not allow evaporating. More malachite will be added by heating the slide over again. Then the slide will be cooled and will be washed under running water. Then, the smear will be flooded with counter stain safranine for 30 second. Then the slide will be washed with distilled water to remove extra stain. The slide will be examined under microscope spores will appear green inside the bacteria while absence of green colour will show non-endospore forming bacteria (Ali et al.,
Test tubes will be poured with about 5.0 ml of the sulfide indole motility medium (SIM) and then it will be autoclaved. The inoculating needle will be flamed and cooled, and it will be inserted into the culture after flaming the neck of the tube. The cap from the tube of the SIM medium will be removed, the neck will be flamed, and it will be stabbed with 2⁄3 way down to the bottom. The neck of the tube will be flamed again before the cap will be returning to the tube. The tubes will be incubated at room temperature for 24 to 48 hours.The SIM cultures will be examined for the presence or absence of a black precipitate along the line of the stab inoculation. The black precipitate of FeS indicates the presence of H2S. (Ali et al.,