STAINING TECHNIQUE 2: DIFFERENTIAL STAINING

DIFFERENTIAL STAINING 

used in identification and classification of bacteria

Gram stain is the most commonly used

Acid-fast stain is used specifically for tubercle bacilli that could not be Gram stained

causative agent of Syphilis

Treponema pallidum

• best examined fresh with the use of dark-field microscope. 

GRAM STAINING TECHNIQUE 

separates bacteria into two groups: (a) Gram-positive bacteria and (b) Gram-negative bacteria. 

gram-positive bacteria

retain a crystal violet-iodine complex through decolorization with alcohol or acetone. appear as purple when viewed by microscopy

gram-negative bacteria

alcohol or acetone removes the crystal violet-iodine complex. These bacteria must therefore be counterstained with a red dye, Safranin, after the decolorization step in order to be visualized by microscopy. Hence, gram-negative bacteria appear as red cells when viewed by microscopy. 

 

GRAM STAINING TECHNIQUE procedure

1. Prepare smear of bacteria from culture. Choose one colony if from culture plates. 

 

2. Stain smear with Crystal violet for 1 minute. 

 

3. Wash off excess stain with tap water, then shake off remaining water. 

 

4. Cover the smear with Iodine solution for 1 minute. 

 

5. Decolorize with 95% ethanol or Gram’s decolorizer for 15-20 seconds. 

 

6. Wash with tap water. 

 

7. Counterstain with safranin red for 30 seconds. 

 

8. Wash off excess stain with tap water. 

 

9. Blot dry or air dry 

 

10. Observe under oil immersion microscope 

 

11. Draw and label your observation 

 

NOTE: Steps 2-8 must be done without pause. Follow the time period prescribed. 

Principles of Gram Staining 

1. The application of the primary stain colors all bacteria blue or violet. 

 

2. Iodine acts as a mordant, binding crystal violet molecules to the bacterial cell wall. 

 

3. 95% ethanol decolorizes the cell if the primary stain is not strongly bind to the cell wall. 

 

4. Safranin counterstains the cells decolorized by 95% ethanol, and does not affect the cells, which retain the primary stain

Sources of False Gram-positive Reaction 

1. Smear was heat-fixed before being air-dried. 

 

2. Smear is too thick. 

 

3. Present of crystal violet sediments. 

 

4. Iodine solution was not thoroughly drained off 

 

5. Decolorize was not left long enough 

ACID-FAST STAIN 

used in staining members of the Mycobacterium species

do not stain easily because of the waxy material present in its wall.

A drastic technique like the use of acid and heat, or use of fast stain like carbol-fuchsin allow staining of this bacteria

Since the bacteria to be examined is highly contagious, prepared slides of phlegm from tuberculous will be examined instead. 

Examination of Prepared Slide  ACID-FAST STAIN 

1. Mount prepared slide of Mycobacterium under oil immersion objective 

 

2. Observe for pinkish or reddish rod-shaped bacteria. 

 

ACID-FAST STAIN  Staining Application (instead of phlegm) 

1. Scrape inner cheek with a toothpick and smear on a clean slide 

 

2. Air-dry then heat-fix. 

 

3. Flood the smear with Carbol-fuchsin and heat the underside of the slide for 5 minutes. Do not allow the stain to boil. 

 

4. Decolorize with acid-alcohol until the smear is faint pink color. 

 

5. Wash the slide with tap water. 

 

6. Counterstain with methylene blue for 30-45 seconds. 

 

7. Wash off excess stain with tap water. 

 

8. Air-dry, then draw and label. 

SPECIAL STAINING 

STRUCTURAL STAINING (SCHAEFFER-FULTON SPORE STAIN) 

CAPSULE STAINING [INDIA INK METHOD] [NEGATIVE STAINING METHOD] 

STRUCTURAL STAINING (SCHAEFFER-FULTON SPORE STAIN) 

utilizes Malachite green to stain the endospore and Safranin to stain the vegetative e portion of the cell

Utilizing this technique, a properly stained spore-former will have a green endospore contained in a pink sporangium

STRUCTURAL STAINING (SCHAEFFER-FULTON SPORE STAIN) procedure

1. Prepare a smear from old cultures of Bacillus subtilis, air-dry and heat fix. 

 

2. Cover the smear with absorbent paper and flood with Malachite green. 

 

3. Steam the slide for 5-10 minutes without reaching boiling point, and keep the smear saturated with Malachite green. 

 

4. Remove the paper, cool the slide and wash with tap water for 30 seconds. 

 

5. Counterstain with Safranin [red] solution for 20 seconds. 

 

6. Wash off excess stain, then air-dry or blot dry. 

 

7. Observe in oil immersion then draw and label. 

CAPSULE STAINING [INDIA INK METHOD] [NEGATIVE STAINING METHOD] 

1. Place a drop of India ink near the edge of a clean slide. 

 

2. Fish a loopful of bacterial culture and mix with the India ink. 

 

3. With a second clean slide at a 30-degree angle touch the mixture and spread it across the width of the first slide. 

 

4. Air-dry the smear. 

 

5. Cover the smear with Safranin red for 30 seconds. 

 

6. Wash off excess stain with tap water. 

 

7. Air-dry or blot-dry, then draw and label. 

Name 5 Gram-positive (Gram+) bacteria

Staphylococcus aureus

Streptococcus pyogene

Bacillus anthracis

Clostridium tetani

name 5 Gram-negative (Gram-) bacteria

Escherichia coli

Salmonella enterica