Theory of Staining Techniques

The purpose of this lab was to identify two unknown bacteria from a mixed culture. The reason for identification of unknown bacteria was to help students recognize different bacteria through different biochemical tests and characteristics. This is important in the medical field because identification of unknown bacteria can help treat a patient by knowing the contributing source of a disease. Also knowledge of different bacteria helped others make antibiotics used today. This lab was completed by using the methods learned thus far in identification of bacteria.

The Gram stain was used to determine if the bacteria was gram positive or negative. A negative test shows a pink color and a positive test is a purple color. When a bacterium is negative it is because it has an outer membrane and a thin layer of peptidoglycan that is much harder to stain. A positive bacterium has a thick layer of peptidoglycan and no outer membrane that can be penetrated by crystal violet.

The next step of the project included preparing a Gram stain to discover the cell shape, arrangement, and if the bacteria is gram positive or

Table 3 shows Gram stain results that indicated C. Freundii as a gram negative bacterium in rod shapes scattered in singles and some in pairs. Each gram stain produced the same results. The Bartholomew and Mittwer method of endospore staining indicated that C. Freundii tested negative for endospore formation. Table 4 shows the biochemical test results of the unknown and the official test results for comparison.

This is important in the medical field because identification of unknown bacteria can help treat a patient by knowing the contributing source of a disease. Also knowledge of different bacteria helped others make antibiotics used today. This lab was completed by using the methods learned thus far in identification of bacteria.

Preliminary studies help identify Genus species of bacteria. Two different preliminary study pathways must be used since two different pathogens were found in the sample. A dilution and a quadrant streak are the ideal methods to separate pure cultures of bacteria. MacConkey Agar and CAN (MAC) is a selective media that is used for the cultivation of gram negative bacteria. (PEA) is a selective media that is used

Two smears of the unknown bacterium #5 were inoculated while the second smear was used for a back up. The unknown bacterium dried for at least forty minutes. After the smears dried, the slides were heat fixed two times to ensure the stability of the organism. The slide was placed on top of the staining rack then over the small sink.

The purpose of this lab was to identify an unknown microorganism using lab techniques. The importance of identifying microorganisms is essential to the survival of humans, expansion of modern day medicine and improvement of quality of life. In 1884, Hans Christian Gram designed a differential staining technique to identify bacteria that would change the future of microbiology. He give rise to a staining process, known as the Gram stain to differentiate microorganisms into two groups between positive and negative gram staining microorganisms. The Gram stain is essential in a lab technique as it distinguishes the cells based on the physical properties of the individual cell walls, and is almost always the first test preformed to differentiate a microorganism. The identification of weather a microorganism is gram positive or negative can revel the bacteria’s virulence, cell wall structure, resistance to antibiotics, resistance to physical disruptions and so much more. In order to identify the unknown provided, unknown #27, the Gram stain was the first test preformed. After discovering that the unknown bacterium was indefinitely a gram positive microorganism, the vast possibilities were narrowed down. However, In order to more definitively identify the unknown, the next step was to preform biochemical tests. A biochemical test identifies metabolic

8. Discuss a possible mechanism of Gram Staining in terms of differences in structure and chemistry between the walls of gram-positive and gram-negative

Figure #1 This picture show the gram stain of unknown #3. The image is under 1000x magnification using oil immersion technique. Gram (-)

Spores can be stained using specific dyes, such as malachite green, that are absorbed by spores in the presence of heat (Noonan).

These are gram positive bacteria which are found in acidic condition of pH 4 to 4.5 – acidophilic.

Starch is a polysaccharide made up of glucose molecules. Some bacteria have an enzyme called amylase which breaks starch down into glucose subunits. The Starch Hydrolysis test is used to determine the production of amylase. Iodine, which is the mordant used in Gram staining, is used in this test to detect the presence of starch. In order to do the Starch Hydrolysis Test I first inoculated a starch plate by using aseptic technique. I streaked the middle of the starch plate with the bacteria into a single line. Then, I let it incubate overnight at 37°C. In week 2, after I added several drops of Gram’s iodine to the starch plate it was ready to be analyzed for starch hydrolysis. The iodine complexes with starch to form a blue-black color in the culture. Clear halos surrounding colonies is the result of their ability to digest the starch due to the presence of amylase.

This report represents my individual effort. I did not receive or offer aid to anyone when performing this assignment, nor did I plagiarize any material.

Bacteria vary greatly in terms of their characteristics and morphology. Colonies can be classified according to their colour, form, elevation, margin and size. Pure cultures of microorganisms