By Momina Shehzadi | BS Zoology Graduate, Pakistan In this article, we will learn Fungi - Morphology, Reproduction, Physiology and Importance - in simple words with diagram. This topic is very important for BS Zoology and Microbiology students. Fungi are found everywhere around us. They are present in soil, in water, in air, on dead plants, on dead animals, on our food, on our skin and even inside our body. When bread becomes old, green or black spots appear on it. These spots are fungi. When we see mushroom growing after rain, that is also a fungus. When yeast is used to make bread soft, that is also a fungus. Fungi are very different from bacteria and plants. They are eukaryotic organisms. It means their cells have a true nucleus and other membrane bound organelles. They do not have chlorophyll, so they cannot make their own food like plants. They get their food from other living or dead things. That is why they are called heterotrophic organisms. The study of fungi is called mycolog...
By Momina Shehzadi | BS Zoology Graduate, Pakistan
In this article, we will learn Endospore Staining - Principle, Procedure, Schaeffer-Fulton Method - in simple words with diagram. This topic is very important for BS Zoology and Microbiology students.
INTRODUCTION
Endospore staining is a special differential staining technique used to visualize bacterial endospores. Endospores are highly resistant, dormant structures formed inside some bacterial cells to survive harsh environmental conditions.
Endospores are not stained by ordinary staining methods like Gram staining because they have thick, impermeable spore coat which prevents entry of dyes. So they appear as unstained clear areas inside vegetative cells in Gram staining.
To stain endospores, special methods with heat are required which force dye to penetrate spore coat. Endospore staining is very important to identify spore forming pathogenic bacteria like Bacillus and Clostridium.
WHAT IS ENDOSPORE
Endospore is thick walled, highly resistant structure formed inside bacterial cell. It is also called spore. It contains bacterial DNA, small amount of cytoplasm, ribosomes and enzymes surrounded by several thick layers.
Endospore formation is called sporulation. It occurs when bacteria face unfavorable conditions like lack of nutrients, high temperature, drying, chemicals or radiation. One vegetative cell forms one endospore. So sporulation is not reproduction, it is survival mechanism.
When favorable conditions return, endospore germinates and forms new vegetative cell. This is called germination.
Bacteria which form endospores are mainly Gram positive rods. Important spore forming bacteria are:
Genus Bacillus: Aerobic spore formers. Example Bacillus anthracis causes anthrax, Bacillus subtilis, Bacillus cereus causes food poisoning.
Genus Clostridium: Anaerobic spore formers. Example Clostridium tetani causes tetanus, Clostridium botulinum causes botulism, Clostridium perfringens causes gas gangrene, Clostridium difficile causes diarrhea.
Endospores are not formed by Gram negative bacteria, cocci or non rod bacteria.
STRUCTURE OF ENDOSPORE
Endospore has complex structure with several layers from outside to inside:
1. Exosporium: Outermost thin protein layer present in some species.
2. Spore Coat: Thick layer made of proteins rich in cysteine, impermeable and resistant to chemicals.
3. Cortex: Thick layer made of peptidoglycan but different from cell wall peptidoglycan, contains calcium dipicolinate.
4. Core or Protoplast: Innermost part containing DNA, ribosomes, enzymes, small amount of water. Core has high amount of calcium dipicolinate which gives heat resistance and low water content which gives resistance to chemicals.
Because of these thick layers and calcium dipicolinate, endospores are highly resistant to heat, drying, radiation, disinfectants and antibiotics. Endospores can survive boiling for hours and can survive for many years in soil.
Location of endospore in vegetative cell:
Central: In middle of cell. Example Bacillus anthracis
Subterminal: Between middle and end. Example Bacillus subtilis
Terminal: At end of cell, giving drumstick appearance. Example Clostridium tetani
Shape of endospore may be round or oval.
PRINCIPLE OF ENDOSPORE STAINING
Principle is based on impermeability of spore coat.
Endospore coat is impermeable to most dyes, so simple stains cannot enter. When primary stain malachite green is applied with heat, heat increases permeability of spore coat and forces malachite green inside endospore. Malachite green is water soluble and has strong affinity for spore material.
Once stained, endospores resist decolorization with water because they retain dye tightly. Vegetative cells are easily decolorized and lose green color.
Then counter stain safranin stains vegetative cells pink or red. So endospores appear green and vegetative cells appear pink.
REQUIREMENTS FOR ENDOSPORE STAINING - SCHAEFFER FULTON METHOD
This is most common method for endospore staining.
Requirements:
Malachite green 0.5 percent as primary stain
Water as decolorizer
Safranin 0.5 percent as counter stain
Bacterial culture with spores - 48 to 72 hours old culture because sporulation occurs in old cultures
Slides, burner, microscope
PROCEDURE OF SCHAEFFER FULTON ENDOSPORE STAINING
Step 1: Preparation of Smear
Make thin smear of bacterial culture on clean slide. Use old culture 2 to 3 days old because young cultures may not have spores. Air dry and heat fix gently. Do not overheat.
Step 2: Primary Staining with Malachite Green with Heat
Flood smear with 0.5 percent malachite green solution. Heat slide from below with small flame until steam rises. Keep steaming for 5 to 6 minutes. Add more stain if it dries. Do not boil. Heating is essential to drive stain inside spore. This step is similar to acid-fast staining where heat acts as mordant.
After steaming, cool slide and wash gently with water. Wash for 30 seconds to remove excess malachite green from vegetative cells. Vegetative cells will be decolorized but spores will retain green color.
Step 3: Counter Staining with Safranin
Flood slide with safranin 0.5 percent for 1 to 2 minutes. Safranin stains decolorized vegetative cells pink.
Step 4: Wash with water, air dry and observe under oil immersion 100X.
Result:
Endospores appear as green or dark green, round or oval bodies inside pink vegetative cells or free outside cells.
Vegetative cells appear pink or red.
OTHER METHODS OF ENDOSPORE STAINING
1. Moeller Method
Uses carbol fuchsin as primary stain with heat and acid alcohol as decolorizer. Spores appear red and vegetative cells blue.
2. Dorner Method
Uses carbol fuchsin with nigrosin as background stain. Spores appear red with dark background.
3. Simple Spore Stain - Using Methylene Blue
Old cultures stained with methylene blue show spores as unstained areas. Quick but not differential.
PRECAUTIONS
1. Use old culture 48 to 72 hours because spores form in stationary phase under nutrient depletion. Young cultures have fewer spores.
2. Do proper steaming for 5 to 6 minutes to ensure stain penetrates spore.
3. Do not overheat to boiling, only steam.
4. Wash adequately with water after primary staining to decolorize vegetative cells properly.
5. Handle spore forming pathogens like Bacillus anthracis and Clostridium tetani carefully because spores are highly resistant and infectious.
IMPORTANCE OF ENDOSPORE STAINING
1. Identification: Helps to identify spore forming bacteria like Bacillus and Clostridium which are important pathogens.
2. Differentiation of Bacillus and Clostridium: Bacillus are aerobic and produce central or subterminal spores without swelling cell. Clostridium are anaerobic and produce terminal spores that swell cell giving drumstick appearance.
3. Sterilization control: Endospores are most resistant forms of life. To check whether autoclave is working properly, strips containing spores of Bacillus stearothermophilus are used as biological indicators. If autoclave kills these highly resistant spores, it means sterilization is effective.
4. Food industry: To detect spore forming spoilage bacteria in canned foods. Clostridium botulinum spores can survive in improperly canned foods and cause botulism which is fatal.
5. Medical importance: Diagnosis of anthrax, tetanus, gas gangrene and botulism requires demonstration of spores.
DIFFERENCE BETWEEN ENDOSPORE AND VEGETATIVE CELL
Vegetative cell is active, metabolizing, sensitive to heat and chemicals, contains more water, stains easily. Endospore is dormant, no metabolism, highly resistant to heat and chemicals, contains less water and calcium dipicolinate, difficult to stain.
RESISTANCE OF ENDOSPORES
Endospores are resistant due to:
Low water content
High calcium dipicolinate
Thick spore coat and cortex
Presence of small acid soluble proteins SASP which protect DNA
Low metabolic activity
They resist boiling at 100 degree Celsius for hours, dry heat, many disinfectants, radiation and antibiotics. They can be killed only by autoclaving at 121 degree Celsius for 15 to 20 minutes or by dry heat at 160 degree Celsius for 2 hours.
CONCLUSION
Endospore staining is special staining technique to demonstrate endospores which are resistant structures for survival. Schaeffer Fulton method using malachite green with heat is most common. Demonstration of endospores helps in identification of Bacillus and Clostridium species and in checking sterilization. For BS Zoology students, this staining is important for practical exams and understanding bacterial survival mechanisms.
FAQs for Students - Endospore Staining
Q1: What is endospore and why it is formed?
Ans: Endospore is highly resistant dormant structure formed inside some bacteria to survive unfavorable conditions like lack of nutrients, heat, drying. It is survival mechanism, not reproduction. One cell forms one spore.
Q2: Why endospores are not stained by Gram staining?
Ans: Because endospores have thick impermeable spore coat which prevents entry of Gram stain. They appear as clear unstained areas inside Gram positive vegetative cells.
Q3: Why heat is used in endospore staining?
Ans: Heat is used as mordant to increase permeability of spore coat and to force primary stain malachite green inside spore. Without heat, stain cannot penetrate thick spore layers.
Q4: What is Schaeffer Fulton method?
Ans: Schaeffer Fulton method is most common endospore staining method using malachite green as primary stain with steam for 5 minutes, water as decolorizer and safranin as counter stain. Spores appear green and vegetative cells pink.
Q5: What is difference between Bacillus and Clostridium spores?
Ans: Bacillus are aerobic, spores are central or subterminal and do not swell cell. Clostridium are anaerobic, spores are terminal and swell cell giving drumstick or tennis racket appearance.
Q6: Why endospores are highly resistant?
Ans: Due to thick spore coat, cortex with peptidoglycan, low water content, high calcium dipicolinate, small acid soluble proteins protecting DNA and dormant metabolism. They resist heat, chemicals, radiation and drying.
Also Read: Acid-Fast Staining - Principle, Procedure and Importance
Written by Momina Shehzadi, BS Zoology Graduate from Pakistan.
---
Comments
Post a Comment