Gram Staining Procedure

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Staining of bacteria forms the foremost and the most important step in the identification of bacteria. Gram staining: differentiates bacteria into two types Gram positive and Gram negative bacteria Gram positive bacteria can be either cocci or bacilli or vibrios. Gram negative bacteria can be either cocci or bacilli.

Motility testing
Motility testing is performed by preparing a wet mount and is then observed under the microscope.
Biochemical tests
The staining will be followed by use of various biochemical reagents and tests to get closer to the identification of bacteria. There are many biochemical tests available for bacterial identification. The biochemical tests that will be used for identification of Bacillus spp. are as mentioned below
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Keep the petri dish cover available. Using a sterile inoculating loop or wooden applicator stick, collect a small amount of bacteria from a well-isolated 18- to 24-hour colony and place it onto the microscope slide. Be careful not to pick up any agar. Using a dropper or Pasteur pipette, place 1 drop of 3% H2O2 onto the bacteria on the microscope slide. Do not mix. Immediately cover the petri dish with a lid to limit aerosols and observe for immediate bubble formation. Observing for the formation of bubbles against a dark background enhances readability.
(b) Coagulase test
The coagulase test differentiates strains of Staphylococcus aureus from other coagulase-negative species. The coagulase test can be performed using two different procedures - Slide test and tube test. For both tests, clumping or clots of any size indicate a positive response.
The slide test is performed by preparing a suspension of bacterial cells mixed into a drop of rabbit plasma on a microscope slide. If bound coagulase is present on the bacterial cells, then the presence of plasma will cause the bacterial cells to clump. The tube coagulase test is performed by mixing bacterial cells into a larger volume of plasma in a small test tube. Formation of a clot will be noted within 24 hours for a positive
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A positive diagnostic test rests on the generation of alkaline by-products of citrate metabolism.
Use a fresh (16- to 18-hour) pure culture as an inoculation source. Pick a single isolated colony and lightly streak the surface of the slant. A needle is the preferred sampling tool in order to limit the amount of cell material transferred to the agar slant. Avoid using liquid cultures as the inoculum source. Citrate utilization requires oxygen and thus screw caps, if used, should be placed loosely on the tube. Incubate at 35oC for 18 to 48 hours. Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium.
Citrate positive: growth will be visible on the slant surface and the medium will be an intense Prussian blue. The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green colour to
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