Gram Staining - Principle, Procedure, and Difference Between Gram Positive and Gram Negative Bacteria

By Momina Shehzadi | BS Zoology Graduate, Pakistan

In this article, we will learn Gram Staining - Principle, Procedure, Reagents and Difference Between Gram Positive and Gram Negative Bacteria - in simple words with diagrams. This topic is very important for BS Zoology and Microbiology students.

Gram Staining 

INTRODUCTION

Gram staining is one of the most important and widely used differential staining techniques in microbiology. It was developed by a Danish bacteriologist Hans Christian Gram in 1884. This technique is used to differentiate bacteria into two large groups: Gram Positive and Gram Negative bacteria. This differentiation is based on the chemical and physical properties of their cell walls. For BS Zoology and Microbiology students, Gram staining is the first and most important practical.

PRINCIPLE OF GRAM STAINING

The principle of Gram staining is based on the structure of the bacterial cell wall. Gram positive bacteria have a thick layer of peptidoglycan (50-90 percent of cell wall) and no outer membrane. Gram negative bacteria have a thin layer of peptidoglycan (10 percent of cell wall) and have an outer membrane containing lipopolysaccharide (LPS).

When primary stain Crystal Violet is applied, both Gram positive and Gram negative bacteria take up the stain. When mordant Iodine is added, it forms a large complex called Crystal Violet-Iodine (CV-I) complex inside the cell.

When decolorizer Alcohol or Acetone is applied, the difference appears. In Gram positive bacteria, the thick peptidoglycan layer gets dehydrated and the pores shrink, so CV-I complex cannot come out. The cells remain purple. In Gram negative bacteria, alcohol dissolves the outer lipid membrane and the thin peptidoglycan layer cannot retain the CV-I complex. The complex is washed out and cells become colorless. When counterstain Safranin is applied, the colorless Gram negative cells take pink or red color, while Gram positive cells remain purple.

REAGENTS REQUIRED FOR GRAM STAINING

1. Primary Stain - Crystal Violet (0.5 percent) - Purple color, stains all bacteria.

2. Mordant - Gram's Iodine - Iodine acts as mordant. It fixes crystal violet inside the cell.

3. Decolorizer - 95 percent Ethyl Alcohol or Acetone-Alcohol - Most critical step.

4. Counterstain - Safranin (0.5 percent) - Red or pink color, stains Gram negative bacteria.

PROCEDURE OF GRAM STAINING - STEP BY STEP

Step 1: Preparation of Bacterial Smear

Take a clean glass slide. Make a thin smear of bacterial culture. Air dry and heat fix by passing slide 2-3 times over flame. Heat fixation kills bacteria and fixes them to slide.

Step 2: Application of Crystal Violet (1 minute)

Flood the smear with Crystal Violet and keep for 1 minute. Wash gently with distilled water. All cells will appear purple.

Step 3: Application of Gram's Iodine (1 minute)

Flood the smear with Gram's Iodine and keep for 1 minute. Wash with water. This is mordant step. Cells still purple.

Step 4: Decolorization (10-20 seconds) - Most Important Step

Add few drops of 95 percent alcohol. Keep for 10-20 seconds only. Do not over-decolorize. Wash immediately with water. This step is critical.

Step 5: Counterstaining with Safranin (30 seconds)

Flood the smear with Safranin and keep for 30 seconds to 1 minute. Wash with water. Blot dry with tissue paper.

Step 6: Microscopic Examination

Observe under microscope using Oil Immersion Lens (100X). Gram positive bacteria will appear purple or violet. Gram negative bacteria will appear pink or red.

DIFFERENCE BETWEEN GRAM POSITIVE AND GRAM NEGATIVE BACTERIA

1. Cell Wall

Gram Positive - Thick (20-80 nm), 50-90 percent peptidoglycan

Gram Negative - Thin (8-10 nm), 10 percent peptidoglycan

2. Outer Membrane

Gram Positive - Absent

Gram Negative - Present, contains LPS

3. Teichoic Acid

Gram Positive - Present

Gram Negative - Absent

4. Lipid Content

Gram Positive - Low (2-5 percent)

Gram Negative - High (20-30 percent)

5. Color After Staining

Gram Positive - Purple or Violet

Gram Negative - Pink or Red

6. Sensitivity to Penicillin

Gram Positive - More sensitive

Gram Negative - Less sensitive

7. Examples

Gram Positive - Staphylococcus aureus, Bacillus subtilis, Streptococcus

Gram Negative - E. coli, Salmonella, Pseudomonas

IMPORTANCE OF GRAM STAINING

1. Clinical Diagnosis - Gram staining helps doctors to quickly decide which antibiotic to give.

2. Classification - It is first step of bacterial identification according to Bergey's Manual.

3. Selection of Antibiotics - Gram positive and Gram negative bacteria have different sensitivity to antibiotics.

4. Food and Water Microbiology - Used to check contamination in food and water.

COMMON ERRORS IN GRAM STAINING

1. Over-decolorization - If alcohol is kept for more than 20 seconds, Gram positive will appear Gram negative.

2. Under-decolorization - If alcohol is kept for less than 10 seconds, Gram negative will appear Gram positive.

3. Old Culture - Old cultures of Gram positive bacteria can become Gram negative. Always use young culture (18-24 hours old).

CONCLUSION

Gram staining is a simple but very powerful tool in microbiology. The difference in color is not just a color difference, it shows fundamental difference in cell wall structure. For BS Zoology students, it is essential to learn not only the procedure but also the principle why Gram positive retains purple color and Gram negative does not.

FAQs for students- GRAM STAINING

Question 1: What is Gram staining?

Answer: Gram staining is a differential staining technique developed by Hans Christian Gram in 1884. It is used to differentiate bacteria into two groups - Gram Positive (purple) and Gram Negative (pink) on the basis of their cell wall structure.

Question 2: Why Gram positive bacteria appear purple and Gram negative appear pink?

Answer: Gram positive bacteria have thick peptidoglycan layer (50-90 percent) which retains Crystal Violet-Iodine complex even after alcohol treatment, so they remain purple. Gram negative have thin peptidoglycan and outer lipid membrane which dissolves in alcohol, so CV-I complex is washed out and they take pink color of Safranin.

Question 3: What are the four reagents used in Gram staining?

Answer: The four reagents are:

1. Crystal Violet - Primary stain

2. Gram's Iodine - Mordant

3. 95 percent Alcohol or Acetone-Alcohol - Decolorizer

4. Safranin - Counterstain

Question 4: What is the most critical step in Gram staining?

Answer: Decolorization with alcohol is the most critical step. If you keep alcohol for more than 20 seconds, Gram positive will also appear Gram negative (false Gram negative). If you keep less than 10 seconds, Gram negative will appear Gram positive (false Gram positive).

Question 5: Why we use young culture (18-24 hours old) for Gram staining?

Answer: Old cultures of Gram positive bacteria lose their ability to retain crystal violet due to damage in cell wall. So they can appear Gram negative. Young cultures give accurate results.

Question 6: What is the difference between Gram positive and Gram negative cell wall?

Answer: Gram positive cell wall is thick, contains 90 percent peptidoglycan, teichoic acid present, no outer membrane. Gram negative cell wall is thin, contains 10 percent peptidoglycan, outer membrane present with LPS, teichoic acid absent.

Question 7: Is Gram staining used in medical diagnosis?

Answer: Yes, it is very important in clinical diagnosis. Doctors can quickly see if infection is caused by Gram positive (e.g., Staphylococcus) or Gram negative (e.g., E. coli) and select antibiotic accordingly before culture report comes.

Question 8: Who invented Gram staining and when?

Answer: Gram staining was invented by Danish bacteriologist Hans Christian Gram in 1884 while working in Berlin.

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Also Read previous complete topic Erythroblastosis

Written by Momina Shehzadi, BS Zoology Graduate from Pakistan.