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 Acid-Fast Staining - Principle, Procedure, Ziehl-Neelsen Staining - in simple words with diagram. This topic is very important for BS Zoology and Microbiology students.
INTRODUCTION
Acid-Fast Staining is an important differential staining technique used in microbiology to differentiate acid-fast bacteria from non acid-fast bacteria. This staining was developed by Paul Ehrlich and later modified by Franz Ziehl and Friedrich Neelsen, so it is also called Ziehl-Neelsen staining or ZN staining.
Acid-fast bacteria are those bacteria which retain primary stain even after washing with strong acid and alcohol. This property is due to presence of high lipid content in their cell wall.
The most important acid-fast bacteria are Mycobacterium tuberculosis which causes tuberculosis and Mycobacterium leprae which causes leprosy. Because these bacteria are very important pathogens, acid-fast staining is routinely used in laboratory for diagnosis.
PRINCIPLE OF ACID-FAST STAINING
Principle is based on composition of bacterial cell wall.
Acid-fast bacteria have thick cell wall containing large amount of lipids, fatty acids and waxes especially mycolic acid. Mycolic acid is a long chain fatty acid with 60 to 90 carbon atoms. This waxy layer makes cell wall impermeable and prevents entry and exit of stains easily.
When carbol fuchsin which is primary stain is applied with heat, it penetrates through waxy cell wall and stains cytoplasm red. The heat acts as mordant and increases permeability of cell wall.
Once stained, acid-fast bacteria resist decolorization with acid-alcohol because mycolic acid layer holds the stain tightly. So they remain red.
Non acid-fast bacteria have thin cell wall with less lipid, so they are easily decolorized by acid-alcohol and lose red color. Then they take counter stain methylene blue and appear blue.
Thus acid-fast bacteria appear red and non acid-fast bacteria appear blue.
REQUIREMENTS FOR ACID-FAST STAINING
1. Bacterial smear of sputum or culture
2. Carbol fuchsin as primary stain - contains basic fuchsin 0.3 gram, phenol 5 ml, alcohol 10 ml, water 95 ml
3. Acid-alcohol as decolorizer - contains 3 percent HCl and 95 percent ethanol
4. Methylene blue as counter stain - 0.3 percent
5. Microscope, slide, burner, staining rack
PROCEDURE OF ZIEHL-NEELSEN ACID-FAST STAINING
Procedure has four main steps: Preparation of smear, Primary staining, Decolorization, Counter staining.
Step 1: Preparation of Smear
Make a thin smear of sputum or bacterial culture on clean glass slide. Air dry and heat fix by passing slide 2 to 3 times over flame. Heat fixing kills bacteria and fixes them to slide.
Step 2: Primary Staining with Carbol Fuchsin
Flood the smear with strong carbol fuchsin solution. Heat the slide from below with small flame until steam rises but do not boil. Keep steaming for 5 minutes. Add more stain if it dries. Do not let stain dry on slide. Heating helps stain to penetrate waxy cell wall. This step is called steaming.
After 5 minutes, cool the slide and wash gently with water.
Step 3: Decolorization with Acid-Alcohol
Flood slide with acid-alcohol decolorizer for 2 to 3 minutes until no more red color comes off. This is most critical step. Over decolorization can make acid-fast bacteria appear false negative, under decolorization can make non acid-fast appear false positive. Wash with water immediately after decolorization.
Step 4: Counter Staining with Methylene Blue
Flood slide with methylene blue counter stain for 1 to 2 minutes. Wash with water, air dry and observe under oil immersion lens at 100X magnification.
Result:
Acid-fast bacteria appear as bright red or pink rods. Example Mycobacterium tuberculosis appears as long slender red rods, beaded appearance, often in groups.
Non acid-fast bacteria, pus cells and background appear blue.
MODIFICATIONS OF ACID-FAST STAINING
1. Cold Method - Kinyoun Method
In this method heating is not required. Higher concentration of carbol fuchsin and phenol is used. It is less effective than hot method but useful for field work.
2. Fluorescent Acid-Fast Staining - Auramine Rhodamine Method
In this method, fluorescent dyes auramine and rhodamine are used instead of carbol fuchsin. Acid-fast bacteria show yellow fluorescence under fluorescent microscope. This method is more sensitive and used for screening of large number of sputum samples.
ACID-FASTNESS IN DIFFERENT BACTERIA
Strongly Acid-Fast: Mycobacterium tuberculosis and Mycobacterium leprae resist decolorization with 20 percent sulfuric acid.
Partially Acid-Fast: Nocardia species resist with 1 percent sulfuric acid but decolorized with stronger acid.
Non Acid-Fast: All other bacteria like E. coli, Staphylococcus are non acid-fast.
There are also acid-fast structures other than bacteria like spores of some protozoa like Cryptosporidium, Isospora and Cyclospora show partial acid-fastness and are stained by modified acid-fast staining with 1 percent sulfuric acid.
IMPORTANCE AND USES OF ACID-FAST STAINING
1. Diagnosis of Tuberculosis: Most important use is to diagnose pulmonary tuberculosis by examining sputum smears. Sputum of TB patient contains acid-fast bacilli AFB. WHO recommends examination of at least two sputum samples.
2. Diagnosis of Leprosy: Skin smears from leprosy lesions are examined for Mycobacterium leprae.
3. Diagnosis of Nocardiosis: Nocardia causes lung and brain infections in immunocompromised patients and is partially acid-fast.
4. Differentiation: Helps to differentiate Mycobacterium from other bacteria because treatment is different.
5. To check quality of disinfection and to identify acid-fast contaminants in laboratory.
PRECAUTIONS
1. Use fresh carbol fuchsin and do not let it precipitate.
2. Do not overheat during steaming, only steam should rise.
3. Do not over decolorize.
4. Use clean new slides.
5. Mycobacterium tuberculosis is highly infectious, so perform staining in biosafety cabinet and take precautions.
COMPARISON OF GRAM STAINING AND ACID-FAST STAINING
Gram staining differentiates bacteria into Gram positive and Gram negative on basis of peptidoglycan. All bacteria are stained by Gram staining. Acid-fast staining differentiates acid-fast and non acid-fast on basis of mycolic acid. Only few bacteria are acid-fast.
Mycobacterium species are Gram positive in structure but they do not stain well with Gram staining because waxy cell wall prevents entry of crystal violet. So they appear as Gram ghost cells. Therefore acid-fast staining is essential for them.
CONCLUSION
Acid-fast staining is a special differential staining technique based on lipid content of cell wall. It is simple, rapid and inexpensive method for diagnosis of tuberculosis which is still one of leading causes of death worldwide. For BS Zoology students, learning principle, procedure and result of acid-fast staining is essential for practical exams and theory.
FAQs for Students - Acid-Fast Staining
Q1: What is acid-fast bacteria?
Ans: Acid-fast bacteria are those bacteria which retain primary stain carbol fuchsin even after decolorization with acid-alcohol due to presence of high amount of mycolic acid in their cell wall. Example Mycobacterium tuberculosis.
Q2: Why heat is used in ZN staining?
Ans: Heat is used as mordant to increase permeability of waxy cell wall rich in mycolic acid and to allow carbol fuchsin to penetrate inside cell. Without heat stain cannot enter acid-fast bacteria.
Q3: What is composition of acid-alcohol decolorizer?
Ans: Acid-alcohol contains 3 percent hydrochloric acid and 95 percent ethanol. In some methods 20 percent sulfuric acid is used. It decolorizes non acid-fast bacteria but not acid-fast.
Q4: What is difference between Gram positive and acid-fast bacteria?
Ans: Gram positive bacteria have thick peptidoglycan and retain crystal violet, appear purple in Gram staining. Acid-fast bacteria have thick waxy lipid layer with mycolic acid and retain carbol fuchsin, appear red in acid-fast staining.
Q5: What is Kinyoun cold method?
Ans: Kinyoun method is cold acid-fast staining without heating. Higher concentration of carbol fuchsin is used. It is less sensitive than Ziehl-Neelsen hot method but easier for routine use.
Q6: Why Mycobacterium tuberculosis is not well stained with Gram staining?
Ans: Because Mycobacterium tuberculosis has thick waxy cell wall with mycolic acid which prevents entry of Gram stain crystal violet. So it appears as colorless ghost cells in Gram staining, therefore acid-fast staining is required.
Also Read: Gram Staining - Principle, Procedure and Importance
Written by Momina Shehzadi, BS Zoology Graduate from Pakistan.
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