Bacterial identification through Gram staining and endospore staining procedures

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.

Colony Description

The three staining procedures, in addition to helping determine certain physiological characteristics of the bacteria in the samples, provided ample opportunity to observe the colony in a directly visual manner, which also assists in the identification process and can provide other useful information, as well. As noted above, the colonies tended to form in a hash-mark pattern, with individual rods or "drumsticks" lying on top of each other at near-right angles, and though sizes varied from sample to sample based on the staining procedures it seemed likely that colonies could form in quite large groups in this manner, perhaps indefinitely large given the proper growth medium. All of these results, from the Gram-positive nature of the bacteria to the appearance of endospores to the shape and arrangement of the bacteria and their colonies, lead to a very likely identification of the bacteria in the sample as Clostridium tetani, the bacteria responsible for releasing the neurotoxin tetanospasmin, responsible for the central nervous condition commonly known as tetanus (Unrein, 2008).

All identified strains of Clostridium tetani -- of which there are eleven -- are Gram-positive and present as endospores at certain stages in their lifecycle (Unrein, 2008). They are also "drumstick" shaped in this endospore state and form the observed patterns described here as "hash-marks," which adds further reliability to this identification (Unrein, 2008). These relatively simple tests are thus stillhighly important in identifying potential sources of diseas.