Lab 5 –Cell Structure and Staining using Microscopy
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NAME Buket Rembert
In Lab 3 you were introduced to microscopy. In this lab you will be adding to that experience by reviewing the differences in cell structure for Prokaryotes and Eukaryotes (previously covered in Lecture Unit #1) and learning about staining of microbes.
Part I. Smear Preparation in Mastering Microbiology.
Log in to MM, go to the Study Area, and then to Microlab Tutor “Smear Preparation”. After viewing the short video, answer the following question: 1. List the major steps in smear preparation
If the slide is not clean, clean the slide.
Label the slide.
Make a dime size circle
Place a drop of saline solution on to the slide.
Sterile the loop/or use a sterile loop and obtain culture of the bacteria from the petri dish or tube. Harvest just enough culture. (only use a tiny amount of culture preparing a smear) Spread the bacteria on the glass slide. Set the slide to air dry. Either heat fix or methanol fix. (do not fix it both ways)
Stain the slides.
Properly dispose of any wastes.(waste methanol, materials contaminated with bacteria, broken glass)
Part II. Atlas Sections
In your Lab Atlas you will need to read Section 5: Bacterial Cellular Morphology and Simple Stains and Section 6: Bacterial Cell Structures and Differential Stains and then answer the following questions: 2. What is the third important feature of microscopy? Why?
Third important feature of microscopy is contrast. To be visible, the specimen must contrast with the background of the microscope field.
3. A simple stain will help determine these 3 features of the specimen on the slide:
Cell morphology, size and arrangement then may be determined.
4-A. What part of the stain is responsible for its COLOR?
Chromophore is responsible for the stain’s color.
4-B. Name 3 examples of basic stains:
Methylene blue, crystal violet, and safranin.
5. List the three things that heat fixing a smear prior to staining achieves: Heat-fixing kills most of the bacteria, makes them adhere to the slide, and coagulate cytoplasmic membranes to make them more visible. It also distorts the cells to some extent.
6. What are the three types of cell morphology in bacteria?
Spheres (cocci, singular coccus)
Rods (bacilli, singular bacillus)
Spirals (spirilla, singular spirillum)
7. Cell arrangement is an important feature that aids in identification of bacteria. What is the difference between staphylococcus and streptococcus?
If the cell continue to divide in the same plane and remain attached, they exhibit a streptococcus arrangement. If the division planes of a coccus are irregular, a cluster of cells is produced to form a staphylococcus.
8. What is the purpose of the negative stain? What example is cited in your Atlas?
The negative staining technique is used to determine morphology and cellular arrangement in bacteria that are too delicate to withstand heat-fixing. A primary example is spirochete.
9. What is the purpose of the Gram stain? What part of the bacterial cells determines the outcome?
The purpose of the Gram Stain is to distinguish Gram-positive and Gram-negative cells. Different cell wall construction of Gram-negative and Gram-positive cells determine the ability to resist decolorization or not.
10. List the 4 steps of the Gram Stain naming the chemical used in each step:
a. Primary stain- crystal violet and.
b. Add iodine as a mordan ( form a crystal violet-iodine complex)
c. Decolorization – ( alcohol or acetone)
d. Counter-stain – Safranin
11. What is the purpose of these differential staining techniques:
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