Name:_______________________________________ Course:____________________

Score:
Date:________________________________________Group No:__________________

GRAM STAINING & ACID-FAST STAINING

Experiment No. 6

Objectives
1. Perform the Gram-staining and Acid-fast staining procedure.
2. Identify the Gram-staining and Acid-fast staining reaction.
3. Distinguish the fundamental shapes of bacteria, and
4. Differentiate the methods used for acid-fast staining.

SCHEMATIC DIAGRAM
 Score:
Name:_______________________________________ Course:____________________
Date:________________________________________Group No:__________________

GRAM STAINING & ACID-FAST STAINING

Experiment No. 6

Objectives
5. Perform the Gram-staining and Acid-fast staining procedure.
6. Identify the Gram-staining and Acid-fast staining reaction.
7. Distinguish the fundamental shapes of bacteria, and
8. Differentiate the methods used for acid-fast staining.
Materials
Glass slide Crystal Violet
Alcohol lamp Gram’s Iodine
Microscope with oil immersion objective Safranin
Inoculating loop Malachite green
Carbol fuschin Methylene blue
Acetone or 95% Acid Alcohol
Sulphuric Acid (hot method)

Guide Questions:

1. Explain why only gram-negative cells undergo decolorization during the Gram staining
procedure.
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

2. How does the Gram staining procedure help the doctor treat patients?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________

3. What is responsible for the acid-fastness of these organisms?

______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
 Score:
Name:_______________________________________ Course:____________________
Date:________________________________________Group No:__________________

GRAM STAINING & ACID-FAST STAINING

Experiment No. 6

4. Why is the Ziehl-Neelsen method called the “hot method” while Kinyoun method the
“cold method?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

5. Why must heat or a surface-active agent be used with application of the primary stain
during acid fast staining?

___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

6. Why must heat or a surface-active agent not required during the application of the
counterstain in acid-fast staining?
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

7. Explain the importance of using methylene blue as the counterstain in the acid-fast
staining method.
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
 Score:
Name:_______________________________________ Course:____________________
Date:________________________________________Group No:__________________

GRAM STAINING & ACID-FAST STAINING

Experiment No. 6
Result
A. Gram Staining

Sample 1 Sample 2
Draw a
representativ
e field

Species: _____________________ Species: _____________________

Cell
Morphology

Shape ____________________________ ____________________________

Arrangement ____________________________ ____________________________

Cell Color ____________________________ ____________________________

Gram
Reaction ____________________________ ____________________________
 Score:
Name:_______________________________________ Course:____________________
Date:________________________________________Group No:__________________

GRAM STAINING & ACID-FAST STAINING

Experiment No. 6

B. Acid-fast staining

Sample 1 Sample 2
Draw a
representativ
e field

Species: _____________________ Species: _____________________

Cell
Morphology

Shape ____________________________ ____________________________

Arrangement ____________________________ ____________________________

Cell Color ____________________________ ____________________________

Acid-fast
reaction ____________________________ ____________________________

References:
 Score:
Name:_______________________________________ Course:____________________
Date:________________________________________Group No:__________________