Staining Techniques Lab Report

4. Describe the test used to detect the presence of each type of biologically important

1) Apply the stain to your first unknown slide and examine it under the microscope.

This experiment was given to us to utilized previous knowledge learned throughout the semester to identify a gram negative unknown bacterium. We had to first learn the difference between a gram negative and a gram positive organism. We started off with doing gram stains to determine whether it was positive or negative. Based on the gram stains, a gram positive stains purple and a gram negative stains pink. A gram positive stains purple because the cell walls is made of a thick peptidoglycan layer and doesn’t

b)What stain might you use to determine whether certain cells had been structurally altered rather than destroyed (e.g. changes in dendritic number, length, etc.)?

Finally, the slide is washed again and stained with a counterstain, such as safranin. The counterstain will dye the gram negative cell and will not effect the gram positive cell.

This stain is helpful and widely employed for any kind of overview staining, modified toluidine blue includes glacial-sulpharic acid, this reagent was used for treating the smears prior to staining, sulphation stage help to clear background material from the preparation by digesting away most of the background material in which almost all background material was removed in both lavages and sputum, and not affect either fungal elements or P. jirovecii enabling the cysts to be seen more clearly, it is specific for cysts and more rapid than silver stain, the disadvantage of modified toluidine blue is the using of noxious chemicals, It may be harmful when inhaled (152).

The purpose of the gram stain test is to see if an organism is gram negative or gram positive based on it’s color and shape. Using an inoculating loop and inserting it into the incinerator for 10 seconds, it was sterilized. A small drop of water was then applied onto a clean glass slide by using the now sterile inoculating loop. The loop was used to spread the organism back and forth on the slide until a good portion of the glass is covered. The organism and water were left to dry completely before heat fixing the glass slide. After heat fixing, over a sink crystal violet was applied to the glass slide for one minute. It was then rinsed with water and iodine was applied for one minute. The slide was then rinsed with water and 95% ethanol was

Indicated by its name, this method of hystogical staining involves two dyes. The first of which is Hematoxylin. As a basic dye, hematoxylin works as an acidophilic substance. Because of this, it stains nucleic acids blue and purple. Thus, with this stain the nuclei of cells are stained blue. The second dye, eosin, is basophilic. Any basic or negatively charged structures, like proteins with cationic amino acid group, are stained pink. Of the two, eosin tends to be the overpowering stain, coloring cytoplasmic content, muscle and connective tissues, erythrocytes, and various other cellular content.

The reaction of the Gram-stain in a bacteria is determined by the biochemical composition within that bacteria. Gram-positive cell walls are composed of tightly linked peptidoglycans which traps the iodine complex, thus retaining the violet color after decolorization is complete. Gram positive,on the other hand, have a

3. Describe the process of how bacteria can be identified by their metabolic products using indicator media.

Gram’s Stain was discovered in 1882, but published in 1884 by a Danish Bacteriologist Doctor by the name of Hans Christian Gram. Gram stumbled upon this method while he was examining some lung tissue from patients that had died of pneumonia. While examining this lung tissue Gram discovered that certain stains were more favorable and retained by the bacterial cells. It was only a few years later that Gram produced a staining procedure that he divided into two groups. The two groups divided almost all bacteria into what he called Gram positive (purple) and Gram negative (pink).

The Gram stain uses crystal violet as a primary stain, ethanol-acetone as a negative stain, and safranin as a counterstain. A small amount of the culture was heat-fixed to a glass microscope slide. The culture was flooded with methylene blue, and allowed to sit for one

Cleaned a microscope slide with a Kem Wipe to avoid bacteria lingering in the air or fingerprints being seen on it, and continued by adding one or two drops o f distilled water on to the slide.

The Gram stain is a chemical preparation which allows bacteria to be classified into Gram positive and Gram negative by their ability to retain the stain. Bacteria which keep the purple stain are Gram positive and those that loose the stain or turn a pink colour are Gram negative. This procedure is based on the differences in the cell walls structure. The bacterial cell walls will all contain peptidoglycan but the gram positive bacteria the peptidoglycan layer will be thicker. Gram negative, as well as having a thinner peptidoglycan layer, will also have lipopolysaccharides covering the peptidoglycan layer causing less of the stain to be retained.

The purpose of this process is to be able to determine the morphology and arrangement of bacterial cells. No heat fixation or other chemicals are done to the slide. After performing my simple stain technique using crystal violet, the morphology of my unknown was bacillus and the arrangement was singly.