The staining procedure for the Hematoxylin and Eosin involved four stages. These stages are removal of paraffin, rehydration, staining and dehydration. Removal of paraffin involved mixing Xylene or Neo-Clear for 3 minutes. It also involved another Xylene or Neo-Clear for 2 minutes. The second stage is the rehydration which involved adding 100% ErOH, 100% ErOH for 1 minute, and 95% ErOH for 1 minute and water for 3 minutes. The staining stage involved mixing Hematoxylin for 2 minutes, and water wash for 2-5 minutes in running water. This stage also involved adding scott solution to blue hematoxylin for 2 minutes. Eosin is also added 1-3 minutes, and this is checked after 1 minute.
The last step was the dehydration which involved adding 1 or 2 dips of 70% ErOH, 1 or 2 dips of 70% ErOH, 1 or 2 dips of 95% ErOH and 1 or 2 dips of 95% ErOH. The rinses were performed quickly to preserve the intensity of the cosin counterstain. After rinsing, 100% ErOH was mixed for 3 minutes twice. In addition, Xylene or Neo-Clear is added for 3 minutes twice. After the four stages were done, the specimen was mounted with Cytoseal XYL and covered with a coverslip before the sample dried.
Difference between reported method and actual method followed
There is a difference between the actual procedures used and the reported method. The actual procedure involved sectioning and staining the specimen (Lung). The specimen had already been fixed and embedded. In sectioning a microtome was used with section material at 5 microns. This was then mounted on slide with albumin as a fixative. In the actual method, the most common stains which were used on lung tissues are hematoxylin and eosin (H& E) and gram stains. The latter stain type served the purpose of establishing whether the lung contained any viral or fungal infections. The H & E staining process was done using a single cryocut for every specimen under observation (Laboratory Handbook, p.1). The process begun with the soaking of the slides that contained the cryocuts in a solution of Mayer’s hemalum. This took a minute. After this, the slides and the cryocuts were washed under running water to the point where they were clear. The slides were then soaked into an eosin solution (1%) for half an hour, they were then dehydrated. Dehydration was done by soaking the slides in three ethanol concentration solutions. These solutions were composed of increasing concentrations, typically 70%, 90% and 100%. The slides took 2 min in each of these solutions. This was followed by soaking of the slides in xylene, a process which took 2 minutes. The slides were then kept in unused xylene till the time of mounting, when they were embedded in Entellan and covered using glass coverslips. After being dried for a minimum of one hour, the specimens were ready for analysis (Laboratory Handbook, p.1).
The presence of bacteria was assessed using gram stains. In gram stain method the tissue on a glass slide was covered with few drops of one of the primary stains. Gentian violet is a mixture of methyl violet and crystal violet. The primary stain rendered all the bacteria uniformly violet. After a minute of exposure to the staining solution, the slide was washed in water. Then is treated with few drop of Gram’s Iodine and allowed to act for a minute. The slide was again washed in water and then decolorized in absolute ethyl alcohol or acetone. The process of decolorization was fairly quick and did not exceed 30 seconds. After the tissue was decolorized, it was washed in water without any delay. The tissue was finally treated with few drops of counterstain such as dilute carbol fuchsin, neutral red or safranin.
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