Pretend you are a teacher preparing a quiz for the text I will provide. 
            Each quiz question should follow the format below. 
            Ensure that you carefully recheck the questions and answers for accuracy. 
            The output should be a ready-to-use list of dictionaries for the Python eval function:

            [

                    "question": "WRITE THE QUESTION HERE",
                    "options": ["A) OPTION_1", "B) OPTION_2", "C) OPTION_3", "D) OPTION_4"],
                    "answer": INDEX_OF_CORRECT_ANSWER_IN_OPTIONS_LIST,
                    "explanation": "EXPLAIN WHY THE ANSWER IS CORRECT"

            ]

            Text:
             to see them in the unstained 
condition. 
 
 Staining techniques 
 Staining combine chemically with bacterial protoplasm that pear (negative charge) 
due to the presence large amount of (Po4-3) 
 * Direct staining- The organism is stained and background is left unstained (basic 
dyes) like (methylen blue) 
 * Negative staining- The background is stained and the organism is left unaltered, 
in this staining the bacterial cells will appear as shiny unstained cells in dark back 
ground(Acidic dyes) like (India ink and Nigrosin stain). 
Stains are classified as: 
 • Simple stain:  
Simple staining is one step method using only one dye.  
Basic dyes are used in direct stain and acidic dye is used in negative stain such as 
crystal violet stain of E.coli. 
 • Differential stain: 
 (Gram Staining, Acid-Fast Staining) 
 • Structural or special stains:  
There are special stains are used to stain the specific parts of bacteria (flagella, 
capsules, granule and spores). 
 
 
Fixed slide preparation: 
 1. Sterile the loop by the flame.   
2. Put a drop of water by loop on slide and mix few bacteria from culture with water 
well until the solution become homogenous.  
3. Spread the solution on the slide.  
4. If the bacterial culture is broth, took loop of bacterial culture directly on the slide 
without using of water.  
5. The slide is dried by air. 
 6. It fixed by flame gently, and the slide is ready to stained bacterial smear 
preparation : 
 1- Gram Stain    
2- Zeihle–Nelseen Stain . 
 
 
 
 
 Gram Staining: 
 
 • Differential Stains use two or more stains and allow the cells to be categorized 
into various groups or types. 
 • Gram staining (or Gram’s method) is an empirical method of differentiating 
bacterial species into two large groups (Gram-positive and Gram-negative)  
• based on the chemical and physical properties of their cell wall. 
 • Gram staining is used to determine gram status to classify bacteria broadly. 
 • It is based on the composition of their cell wall. 
 • Gram staining uses crystal violet, to stain cell walls, iodine as a mordant, and a 
counterstain fuchsine or safranin, to mark all bacteria 
 A-Gram positive bacteria: If they retain the primary dye complex of crystal violet 
and iodine (purple or blue color). 
 B-Gram negative bacteria: If the decolorization occurs and bacterial cells accept 
the counter stain. (red color)
 
Procedure of  gram  stain: 
• Add primary stain /crystal violet stain (purple or violet color)  on fixed smear for 
1-2 minute. 
 • Wash with water  
• Add iodine solution (brown color ) for 1 minute. 
 •Wash with water  
• Add decolorizer 
 • (acetone alcohol) (colorless) Wash with water for 15 second. 
 • Add counter stain/ safranin stain (red or pink color) for 1-2 minute. 
 •Wash with water  
• Dry the slide with air. 
 • Examine the slide with microscope by using 4x ,10x, 40x, and 100x . 
 
 
 
 
The results: 
 • Gram positive bacteria appear with purple or violet color due to crystal violet and

            Quiz: