Name and course section: LeKesha Hinds
Title: Bacterial Morphology- Lab #2
Purpose: Being able to observe morphologies by preparing wet mount slides and learning direct and indirect staining techniques.
Procedure: First, set up the microscope. View the prepared slides of bacterial morphology. Use each morphological type as a comparative tool for the remainder of the exercise. Disinfect your work area with a 10%-bleach solution using the Procedures in the Preparation of Disinfecting Solution section in the Appendix. Use the marker pencil to make a dime-sized circle on each of the three slides. Use a clean pipet to add a drop of warm water to the circle on the first slide. With the cotton swab vigorously scrape the inside of your mouth and gums. Smear the swab inside the circle on the first slide, transferring as much material to the drop of water as possible. Cover the drop with a cover slip. Use the pipet to add a drop of water to the circle on the second slide. Use the toothpick to scrape a sample of plaque from your teeth.
Transfer the plaque from the toothpick to the drop of water, mixing well to dissolve any clumps. Cover the drop with a cover slip. Use the pipet to add a drop of the S. cerevisiae mixture to the circle of the third slide. Cover the drop with a cover slip. Set the cup of yeast mixture and its pipet aside for later use.

Second, do the same as for the first slides. When the slides are completely dry, heat-fix each slide with a flame source. Hold sample side up, with a clothespin or test tube holder. Leisurely pass the slide over the flame 3–4 times. But too much heat may distort the organism. Keep the slide out of the direct flame but close to the heat. The slide should feel very warm but not too hot to hold. Place the first slide in the staining tray. Add a drop or two of Gram Stain Solution #1, crystal violet, to the slide to cover the sample. Allowing the crystal violet to sit on the slide for 30 seconds. Then gently rinse the slide with water. Blot the slide dry with a paper towel. Repeat steps 7-9 for the remaining slides.

Third, do the same as 1 and 2. Place a small drop of Congo red on the side of slide #1. Use a pipet to add a drop of yeast culture to the drop of Congo red. Place a clean cover slip over the preparation. Press the slip down and blot gently with a paper towel to get a thin, even film under the cover slip. Set the slide aside. Place a small drop of Congo red on slide #2. Place a drop of Congo red on the side of slide # 3. Mix 1 tablespoon of bleach into the yeast culture and let it stand for at least 30 minutes to ensure all organisms have been destroyed. Then discard the contents. Clean your slides and disinfect your area thoroughly with a 10%-bleach solution.

Data Table:
Exercise 1: Prepared Slides Prepared slides | 10X | 40X | 100X | Cocci | Hard to see | Tiny cells | Able to see nucleus in the center | Bacillus | Tiny dot | Dots looking more like a shape | Tiny rods | Spirillum | Tiny black spots | Black lines | Spiral shaped cells with visible nucleus |
Exercise 2: Wet Mount Preparations Preparation | 10x | 40x | 100x | Mouth Swab | Several shaped cells | Varied different cells | | Plaque | | Dark circular shapes | | Yeast | | Odd shaped | Very distinct edges |
Exercise 3: Direct Staining Preparation | 10x | 40x | 100x | Mouth Swab | two clusters that were fairly easy to recognize | All of the cells were cocci. | Some of the cells were large others were nonexistent. | Plaque | different layers of cells | cells were rectangular in shape and varied in size | nucleus was visible in each cell | Yeast | Different shapes | Nothing | Nothing |
Exercise 4: Indirect Staining Preparation | 10x | 40x | 100x | Mouth Swab | Visible | Can see cocci and bracillus | Length varied | Plaque | Clearer | cells were rectangular in shape and varied in size | nucleus was visible in each cell | Yeast | Different shapes | Nothing | Nothing | Results: I was able to observe morphologies in my prepared wet mount slides and learned how to correctly use direct and indirect staining.

Questions: A. List three reasons why you might choose to stain a particular slide rather than view it as a wet-mount. 1. Provides a reliable means for observing bacteria. 2. Bacteria have almost the same refractive index as water. Straining cells makes them easier to see. 3. Allows bacteria morphology to be visualized easily.

B. Define the following terms:
Chromophore: a group of atoms within a molecule which are responsible for the color of the molecule. Chromophores are found in a wide variety of molecules, and can work in several different ways. Groupings of atoms within a molecule which contribute in some way to its characteristics are also known as moieties or functional groups; a chromophore adds color to a molecule because of the nature of the atoms involved and the way they are bonded with each other.
Acidic Dye: dyes that ionize in solution to produce negatively charged ions or anions; they consist of sodium salts of phenols and carboxylic acid dyes; their solutions tend to be neutral or slightly alkaline; examples are eosin and aniline blue.
Basic Dye: any colored substance containing auxochromes and thus capable of coloring substances to which it is applied; used for staining and coloring, as a test reagent, and as a therapeutic agent.

C. What is the difference between direct and indirect staining?
Direct staining stained bacteria with basic dye (methylene blue, carbol fuchsin, crystal violet,etc) which its colouring power is in positive ion, and reacts with the negatively charged cell. Thus, the bacterial cell will be stained with the basic dye's colour. In other words, direct staining can be explained as staining the bacterial cell. Indirect staining stained bacteria with acidic dye (nigrosine, sodium eosinate,etc) which its colouring power is in negative ion. Since bacteria cell is in negative charged ion, thus an acidic dye does not stain with negatively charged bacterial cell. Instead, it forms a deposit around the cell. Indirect staining can be explained as staining the background of the bacterial cells leaving the cell colourless. The indirect staining gives a more accurate view of the bacterial cell, clearer picture of its size and shape.

D. What is heat fixing?
It helps the cells adhere to the slide so that they can be stained. The purpose of heat fixing is to kill the organisms without serious distortion. They adhere better to the slide and also take up dye more easily. E. Why is it necessary to ensure that your specimens are completely air dried prior to heat fixing?
To allow the excess water to dry out before heating. If heated right after, the water would cause the smear to overheat and denature some features in the stain, and those features would no longer be seen. F. Describe what you observed in your plaque smear wet-mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences?
G. Describe what you observed in your cheek smear wet-mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences? The cheek wet mount had several shapes and different cells. The direct stain had two clusters that I could see and a nucleus in the center. The indirect cell was visible. Was able to see cocci and bracillus and the nucleus cell.

G. Describe what you observed in your yeast wet-mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences? Yeast wet mount had odd shapes, but was hard to see. The direct stained and indirect stained had different shapes, but I couldn’t see anything else.

I. Were the cell types the same in all three specimen sets: yeast, plaque, and cheek? How were they similar? How were they different?
No, there shapes and sizes were different.