Lab 5 Cell Structure And Staining Using Microscopy 1

Lab 5 –Cell Structure and Staining using Microscopy

Instructions: Please download this MSWord document to your computer and answer the questions as asked. Then save the document and upload it to Bb using the Assignment feature provided. This assignment is worth a total of 100 points – there are 20 questions worth 5 points each.

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: a. Acid-fast It is used as a differential stain to detect cells capable of retaining a primary stain when treated with an acid alcohol. It is important differential stain used to identify bacteria in the genus Mycobacterium., coccidian parasites, acid-fast bacilli.

b. Capsule Stain It is a differential stain used to detect cells capable of producing an extracellular capsule. Capsule production increases virulence in some microbes such as the anthrax bacillus and the pneumococcus.

c. Endospore Stain The spore stain is a differential stain used to detect the presence and location of spores in bacterial cells. Only a few genera produces spores.

Part III. Staining in Mastering Microbiology.
Returning to Mastering Microbiology, go to the Study Area, Microlab Tutor and view the video on “Gram Staining”. Then go to “Microbiology Animations with Quizzes” and work your way through “Staining”. Then answer the following questions by circling the letter of the correct answer:
12. Which type of staining method would you use to determine endospore-forming cells from non-endospore-forming cells? a. Specialized b. Differential c. Regular d. Simple

Answer is D

13. Which of the following is a characteristic of simple stains? a. They stain specific structures of a bacterial cell. b. They stain specific structures of a bacterial cell AND can be rinsed with water. c. They can be rinsed with water. d. They are a basic stain. e. They are a basic stain AND can be rinsed with water.

Answer is E

Part IV. Structure and Microscopy – Lab 5 from eScience Lab Manual.
Read the background information on p. 76-79. Following the instructions, prepare your Agar Plate and inoculate it with specimens from two different surfaces as directed.
14. Photograph of your plate after inoculation and incubation. You may insert your photograph here or you may upload it as separate attachment in Bb.

15. What 2 surfaces did you swab for your samples? Which sample provided the most growth?

I wanted to take this opportunity and try different surfaces. I have swabbed my trashcan and my cellphone. I was expecting trashcan to have higher bacteria. Both of the surfaces I swabbed did not end up the amount of growth I expected. I have observed 3 different types of colonies growing on the trashcan though. (My incubation time was 6 days)

Part V. To simulate the Gram Stain process, go the website listed on Bb for the “Virtual Gram Stain.”
To run the process, click on “View Example” and then start by clicking on the 1st Test Tube to be used in the Gram Stain process. For each subsequent step, click on the next tube to be used in the process.

I have completed this fun little practice. It helped to clarify the staining procedure.

Part VI. Returning to the eScience Lab Manual, prepare your three slides as directed on p. 81-83. In the absence of a microscope with which to view your slides, refer to the color photographs of bacteria in Section 5 and 6 in your Lab Atlas.

16. Photograph your slides and insert the clearly labeled photographs or upload them as separate attachments on Bb.

17. In the Gram Stain, what different characteristic(s) exist between the two groups that allow for the different staining conditions?

The cell envelopes of most bacteria fall into one of two major groups. Gram-negative bacteria are surrounded by a thin peptidoglycan cell wall, which itself is surrounded by an outer membrane containing lipopolysaccharide. Gram-positive bacteria lack an outer membrane but are surrounded by layers of peptidoglycan many times thicker than is found in the Gram-negatives.

18. Why was the Gram iodine added to the Gram staining technique?

Gram iodine is added to the Gram staining as a mordant to enhance crystal violet staining.

19. Why is a counterstain (safranin) added to the Gram staining procedure?

After decolorization procedure, Gram-positive cells remain purple whereas Gram-negative cells colorized by the counterstain Safranin. Upon successful completion of a Gram stain, Gram-positive cells appear purple and Gram –negative cells appear reddish-pink.

20. What are the advantages of performing a negative stain versus a simple stain for visualizing bacteria?

Even too delicate bacteria can be treated with negative stain. Also when the accurate size is crucial, a negative stain can be used because it produces minimal cell shrinkage. It provides a more detailed assessment of a microbe’s morphology than simple staining does because beckground rather than the microbe is stained. The microbe’s ultrastructure is preserved and it outlines the cells and makes it highly visible.