Questions 1. Describe the major differences between gram positive and gram negative bacteria cell walls.

The gram negative bacteria cell wall is a thin peptidoglycan layer and an outer cell membrane with a lipopolysaccharide layer. The gram positive bacteria cell wall is a single thick peptidoglycan layer. This wall forms in a mesh like formation of three layers of alternating material.

2. From the procedure that you have carried out,do you feel that the Gram positive stain
is a simple procedure? No, because there are many procedure to do and more complex than simple ones and use more than one stain to differentiate cellular components. Discussion The Gram stain procedure uses 3 different stains. These are crystal violet, Grams iodine, and safranin. The cells are first stained with crystal violet, then Grams iodine. Following a rinse in alcohol, to de-colorize the cells, the cells are then stained with safranin. The Gram stain procedure separates almost all bacteria into two large groups: the Grampositive bacteria that stain blue and the Gram-negative bacteria that stain pink. Bacteria take up the Gram stain differently because they differ in cell wall composition. Gram-positive bacteria have a thick cell wall layer. Alcohol does not readily penetrate to decolorize the cell wall of the previously applied crystal violet stain. Gram-negative cells have a thinner cell wall through which the alcohol readily penetrates. The crystal violet is removed from these cell walls that are then stained with the safranin counterstain. Staphylococcus aureus, and Bacillus subtillis are Gram-positive and stain blue. E. coli is Gram-negative and stain pink. Conclusion Differential stains are more complex than simple ones and use more than one stain to differentiate cellular components. They are used to examine structural differences between bacterial groups or to provide contrast to different structures within the same organism.

Preparation Of Films For Staining Title: preparation of films for staining Objective: to produce a thin smear of bacteria adhering to a clean microscope slide as preparatory to staining. Procedure: A. From broth cultures 1. Inoculating loop was sterilized using Bunsen burner and let to be cool before use it to obtain bacterial suspension from the tube. 2. The bacterial then placed in the center of the clean microscope slide. It also speared to produce thin films. 3. The films are set to air dry before wafting the slide gently on the Bunsen flame to fix and kill pathogenic bacteria. 4. The slide was place on a rack then the stain were apply. B. From agar cultures. 1. A drop of water was place in the center of the glass slide. 2. With sterilized inoculating loop to obtain a minute of bacterial culture from the agar cultures 3. It then mix with the drop of water and then proceeds as broth cultures. SIMPLE STAINING TECHNIQUES Material: 1. 24 hours broth cultures of a. E. coli b. Staph. aureus 2. 24 hours nutrient agar slants of a. Bacillus subtilis b. Pseudomonas aeruginosa 3. Slides 4. Inoculating loop 5. Dye solution a. Crystal violet b. Methylene blue c. Carbol fuchsin 6. Test tubes Procedure: 1. By using inoculating loop a minute of bacterial was transferred onto the glass slide. 2. Then a film of slide was prepared. 3. The prepared film then flooded with crystal violet, methylene blue,and carbol fuchsin. All of the stain it let to react about 30 seconds. 4. The excess stained was wash with slow running water. The excess water then blot before dry with Bunsen flame. 5. The slide then examine without the cover slip under the objective lens (x100) 6. The observation then recorded.

Question 1. In preparing a slide from a colony growing on an agar medium, why is it necessary to place only a minute portion of the colony on the slide? To obtain a good slide from a colony growing on an agar medium it is necessary to place only a minute portion of the colony on the slide. Discussion From the experiment,a simple stain consists of a solution of a single dye. Some of the most commonly used dyes are methylene blue, carbol fuchsin, and crystal violet. Simple stains allow one to distinguish the shape of the bacteria. For example, E. coli and Bacillus Subtillus are bacilli or rod-shaped bacteria. Many bacilli occur singularly, but chains may also be observed. Bacilli very greatly in length and diameter.Staphylococcus aureus is cocci or spherical bacteria. Cocci may occur singularly.

Conclusion A simple stain employs a single stain that is used primarily to examine shape and arrangement of cells. Basic stains such as methylene blue, crystal violet, or carbolfuchsin, are often used for this type of staining because they will react with negatively-charged molecules in the cell,example, nucleic acids and cell wall components.