2420-Lab 3- Bacterial Staining Techniques-II-(Differential)

Directions:

Answer following questions after reading the information and watching the video from the link below. Use color RED or BLUE for your answers. Submit the completed document on eCampus for grading. Refer to divulge

· the textbook chapter 3 (3.2) (Nester- McGraw Hill)

· Link: Bacterial Staining Techniques-II, Virtual Edge Experiment-4

Differential Staining

1. What is differential staining?

Differential staining is the process of staining that involves the use of a single stain. This allows for efficient differentiation of structures/cellular components, and microorganisms of a single organism.

Gram Staining (4A on Virtual Edge)

Watch the video of Gram Staining from the textbook chapter 3- review 3.2 (pages 53-54), 3.5 (pages 65-67– cell wall of prokaryotic cells) and answer the following questions.

2. What is the purpose of Gram staining?

Gram staining allows for the determination of chemical composition of bacterium cell walls.

3. Who invented the Gram staining procedure?

Hans Christian Gram

4. What is the first stain used in Gram staining procedure?

The basic dye crystal violet (primary stain)

5. Do all cells pick up this stain?

Bacteria cell walls pick up the primary stain (crystal violet).

6. What is the function of a mordant?

A mordant chemically alters the shape of the dye molecule. This allows for the intensification of the stains in the cells/tissue preparations.

7. Which mordant is used in the Gram staining procedure?

Iodine

8. What would be the result if the mordant is forgotten from the procedure?

The crystal violet molecules will not form a complex the reaction will be red.

9. What is alcohol used for?

The alcohol is a lipid solvent used to dissolve the gram’s-negative cell wall outer membrane. Alcohol acts as the decolorizing agent.

10. Why is decolorization necessary?

Allows for the differentiation of the gram-positive bacteria from the gram-negative bacteria

11. What is a counterstain?

This is a stain with contrasting color to the principal stain. This increases visibility under the microscope.

12. Which counterstain is used in Gram staining?

Safranin

13. What are cells called that retain the primary stain?

Gram positive

14. What are cells called that lose the primary stain?

Gram negative

15. What are cells called that take up the counterstain?

Gram positive and gram negative

16. What type of cells do not take up the counterstain?

Gram positive

17. After the Gram staining procedure, Gram positive bacterial cells appear ___purple_______ colored.

18. After the Gram staining procedure, Gram negative bacterial cells appear _____red_______ colored.

19. Which component of the cell wall in Gram positive bacteria is responsible for the retention of the primary stain?

Peptidoglycan

20. Which component of the cell wall in Gram negative bacteria is responsible for the retention of the counterstain?

Peptidoglycan

21. What would be the color of Gram negative bacterial cells if the decolorization step is forgotten? Explain.

The color would remain purple. This will be regarded as a false positive result.

22. Which bacteria were used in this experiment and what were their shapes? (From the textbook, review 3.3- Morphology of prokaryotic cells)

Spherical in shape. The cells used were prokaryotic cells.

23. From what you have learned from the textbook, if a patient has E. coli infection which are Gram negative bacteria, would you advise them to take a course of Penicillin? Why or Why not?- Explain

No, it is not advisable for the patient to take penicillin. This is because E. coli is resistant to beta lactams like penicillin and other non-beta lactams.

24. Following picture shows the Gram staining result. Point out (or describe) Gram positive and Gram negative cells from the picture. Be sure to specify morphology:

The gram positive cells are the violet/purple looking cells under the microscope. The gram positive cells appear purple because the bacteria will retain the primary stain i.e. crystal violet. The gram negative bacteria will appear pink under the microscope.

Acid-fast Staining- (4B on Virtual Edge):

Read the text from the following to help answering the following questions.

· link Acid-fast Staining,

· textbook chapter 3- section 3.2

25. List two genera of bacteria that are stained for identification by using Acid-fast staining method.

Nocardia species, mycobacterium avium-intracellulare complex

26. Which fatty acid is found in the cell walls of acid-fast bacteria?

Mycolic acids

27. Which primary stain is used in the acid-fast staining procedure?

carbol fuchsin

28. Why is steaming necessary at this step?

Steaming loosens up the waxy layer hence promoting entrance of the primary stain in the cell.

29. Which counterstain is used in the acid-fast staining procedure?

Methylene blue

30. Why is acid-alcohol used to decolorize in the acid-fast staining process instead of regular ethanol?

Acid-alcohol acts as the decolorizing agent because the acid-fast cells are resistant to decolorization because the primary statin is soluble in the cellular waxes as compared to the decolorizing agent.

31. Which two types of bacteria were used in this experiment?

Nocardia species, mycobacterium avium-intracellulare complex

32. Which of the above two types of bacteria were acid-fast? (or retained the primary stain?)

Mycobacterium retains the primary pink stain.

33. From the picture below, point out the acid-fast cells:

The red cells in the image s

34. If you are working in a clinical diagnostic laboratory, which of the above staining methods would you use to predict the cause of infection under following conditions?

a. Tuberculosis

Sputum smear microscopy

b. Strep throat

Gram staining

 

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