Gram Stain Bacteria Identification Following Standard Procedure

Gram Stain

Bacteria Identification

Following standard procedure for Gram staining, a slide was prepared by heat fixing the sample and applying the primary crystal violet stain (Bruckner, 2012). After incubation in the primary stain for a period of one minute, the stain was rinsed under a slow stream of water for four seconds before fixing the remaining dye with Gram's iodine, with immersion in this mordant lasting one minute (Bruckner, 2012). Following this, a second rinse using acetone, again poured in a very slow stream and this time lasting for less than three seconds, removed any non-fixed crystal violet and left behind only the dye fixed within the Gram-positive bacteria of the sample (Carleton, 2012). Safranin was added as a secondary stain and incubation again lasted one minute, with a rinse of four seconds under a slow stream of water occurring before examination of the slide (Carleton, 2012). All observable bacteria were dyed violet, meaning the sample was of a Gram-positive species (Bruckner, 2012). The bacteria were arranged in a hash-mark pattern noticeable in larger gatherings of cells, though definition of these groups and of individual bacteria was not especially high.

Endospore Stain

A sample slide was prepared and heat fixed, then placed on wire gauze in a ring stand and absorbent paper laid directly on the sample (Rossbach, 2012). The paper was saturated with malachite green and the slide then heated with a Bunsen burner until steaming (Rossbach, 2012). Dye and heat were added alternately just to the point of steaming for the next three minutes, with heat removed as soon as steaming occurred and dye added only when the paper was dry, taking care not to over-saturate or overheat the sample (Rossbach, 2012). The paper was then removed and the slide rinsed for twenty seconds under a steady stream of warm water, and a counter stain of safranin was applied in an immersion lasting forty five seconds (Rossbach, 2012). Endosporic cells in this staining procedure retain the green malachite dye, while other organic matter retains only the red safranin counterstain; observation of the stained slide revealed only green structures, in a similar arrangement as observed in the Gram stain procedure, suggesting a Gram-positive endosporic bacteria.

Negative Stain

A small drop of nigrosin stain was placed to one side of a cleaned and flamed slide, and a small loop of the bacteria was placed in the center of the dye pool (Rossbach, 2012). A second cleaned and flamed slide was used to spread the dye and culture mixture carefully and evenly along the sample slide, resulting in a thin and even coating of dye/culture along the plan of the original sample slide (Rossbach, 2012). No heat was applied, but the dye was allowed to dry at room temperature, which took approximately five minutes in experimental conditions; after drying, the sample was ready for observation without any additional rinsing or counterstaining, with the background of the slide appearing the grey color of the dye and the un-dyed bacteria sharply outlined against this grey backdrop (Rossbach, 2012). The bacteria appeared in a similar formation to that observed in the previous two staining procedures, however with far greater clarity -- the individuals rods of the bacteria that made up the hash-mark pattern observed in the Gram stain could be distinguished here.

Colony Description