Types of staining- Principle and procedure

Gram staining, Acid-fast staining; staining of capsule, cell wall, endospore, inclusion bodies

1. Types of staining Gram staining, Acid-fast staining; staining of capsule, cell wall, endospore, inclusion bodies

2. Acid Fast Stain/ Ziehl-Neelsen stain Acid-fast mycobacteria contain mycolic acid in their outer membrane, making the cells waxy and resistant to staining with aqueous based stains such as the Gram stain. The primary stain, carbolfuchsin is applied to the cells, and heat and phenol are used to allow the stain to penetrate into the waxy surface of acid-fast microorganisms. The excess stain is removed with treatment by acid alcohol (ethanol and hydrochloric acid). A secondary stain, methylene blue, is then applied to the cells . Primary Stain:  0.3% Carbol-fuchsin . Dissolve 50 g phenol in 100 mL ethanol (95%) or methanol (95%). Dissolve 3 g Basic fuchsin in the mixture and add distilled water to bring the volume to 1L. Decolorization Solution:  Add 30 mL hydrochloric acid to 1 L of 95% denatured alcohol. Cool and mix well before use. Alternate decolorizing reagent (without alcohol): Slowly add 250 mL sulfuric acid (at least 95%) to 750 mL distilled water. Cool and mix well before using. Counterstain :  0.3% nethylene blue. Dissolve 3 g nethylene blue in 1 L distilled water.

3. Preparation of microscope slide: Acid-fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids. These acids resist staining by ordinary methods such as a Gram stain. It can also be used to stain a few other bacteria, such as Nocardia . Clean slide with a Kimwipe and alcohol to remove any fingerprints. Draw two circles with your Sharpie on the  bottom  of the slide. Using your inoculation loop, put two  small  drops of water in each circle. Using aseptic technique, remove a  very small  amount of bacteria from the culture tube. Make sure you flame the tube before and after you enter. Smear the bacteria in the drop of water on your slide. You may go out of the perimeter of your circles! Let the slide  air dry completely . Heat-fix the slide by running it through the flame 3-4 times with the ‘smear’ side up. Do not flame the side with the bacteria! Let the slide cool completely and you are ready to stain it .

4. Staining procedure: Cover the smears with a piece of paper towel within the border of the slide. Place the slide over a beaker of steaming water. Do not let the beaker boil dry Flood the paper towel with  carbolfuchsin  and let the slide steam for 3-5 minutes. Complete the rest of the procedure at the back sinks. Remove the stained paper towel and discard it in the trash can, not in the sinks. Gently rinse the slide with water to remove any pieces of a loose paper towel and tap dry. Apply  acid-alcohol  for 15-30 seconds. Rinse off and tap dry. Counterstain with  methylene blue  for 1.5 minutes. Rinse with water and tap dry. Blot gently with bibulous paper. Dry the bottom of the slide before placing it on the stage of the microscope and view with the oil immersion lens.

5. INTERPRETATION Acid-fast staining is a laboratory test that identifies the presence of acid-fast bacteria (AFB) in a sample of tissue, blood, or other body fluid. AFB are a group of bacteria that have a unique physical property called acid fastness, which allows them to resist decolorization by acids during staining.  Here are some things to consider when interpreting the results of an acid-fast stain:  Presence of AFB The presence of AFB indicates a possible mycobacterial infection, but it's not specific to tuberculosis (TB).  Sensitivity Acid-fast staining may not be sensitive enough to detect TB organisms in smears or Cytospin preparations. A negative result doesn't rule out infection.  Cell structure AFB have a gram-positive cell envelope with a high lipid content and a waxy-like cell wall that's difficult to penetrate.  Organisms Some examples of AFB include:  Mycobacterium : M. leprae , M. tuberculosis, M. smegmatis , M. Avium complex, and M. kansasii   Nocardia : N. brasiliensis , N. cyriacigeorgica , N. farcinica , and N. nova  

6. Capsule Staining The main purpose of capsule stain is to distinguish capsular material from the bacterial cell. A  capsule  is a gelatinous outer layer secreted by bacterial cell and that surrounds and adheres to the cell wall. Most capsules are composed of polysaccharides, but some are composed of polypeptides. The  capsule  differs from the  slime layer  that most bacterial cells produce in that it is a thick, detectable, discrete layer outside the cell wall. The capsule stain employs an acidic stain and a basic stain to detect capsule production. Principle of Capsule Staining Capsules stain very poorly with reagents used in simple staining and a capsule stain can be, depending on the method, a misnomer because the capsule may or may not be stained. Negative staining methods contrast a translucent, darker colored, background with stained cells but an unstained capsule. The background is formed with  india ink or nigrosin or congo red. India ink is difficult to obtain nowadays; however,  nigrosin is easily acquired. A positive capsule stain requires a mordant that precipitates the capsule. By counterstaining with dyes like crystal violet or methylene blue, bacterial cell wall takes up the dye. Capsules appear colorless with stained cells against dark background. Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by heating. A drop of serum can be used during smearing to enhance the size of the capsule and make it more easily observed with a typical compound light microscope.

7. Procedure Place a small drop of a  negative stain  (India Ink, Congo Red, Nigrosin , or Eosin) on the slide. Congo Red  is easier to see, but it does not work well with some strains.  India Ink  generally works, but it has tiny particles that display Brownian motion that must be differentiated from your bacteria.  Nigrosin  may need to be kept very thin or diluted. Using sterile technique, add a  loopful of bacterial culture to slide, smearing it in the dye. Use the other slide to drag the ink-cell mixture into a thin film along the first slide and let stand for  5-7 minutes. Allow to air dry (do not heat fix). Flood the smear with  crystal violet stain  (this will stain the cells but not the capsules) for about  1 minutes . Drain the crystal violet by tilting the slide at a 45 degree angle and let stain run off until it air dries . Examine the smear microscopically (100X) for the presence of encapsulated cells as indicated by clear zones surrounding the cells.

8. Result and interpretation Capsule:  Clear halos zone against dark background No Capsule:  No Clear halos zone Examples of Capsule Positive and Negative Positive Bacillus anthracis , Klebsiella pneumoniae , Streptococcus pneumonia  Neisseria  meningitidis  Clostridium  spp ,  Rhizaobium   spp , etc. Negative Neisseria gonorrhoreae ,  etc. S treptococcus pneumoniae K lebsiella pneumoniae H aemophilus influenzae P seudomonas aeruginosa N eisseria meningitidis C ryptococcus neoformans

9. interpretation Capsule staining is a technique used to identify the presence of a capsule around a bacterial cell or yeast, and the interpretation of the results is as follows:  Capsule present:  In a capsule stained image, the capsule appears as a clear halo around the cell, against a darkly stained background. The bacterial cells are usually stained purple.  Capsule absent : If no capsule is present, the cell will appear stained.       Capsule staining is important because the presence of a capsule is related to a microbe's virulence, or ability to cause disease. The capsule is a gelatinous outer layer that surrounds and adheres to the cell wall. It's often difficult to see with standard staining procedures because the capsule is water-soluble.  Capsule staining uses a negative staining technique, where the dye stains the background but not the capsule. This is because capsules don't absorb most basic dyes. A common negative staining technique is to add a few drops of India ink or nigrosin to the specimen.  You can also combine positive and negative staining techniques to visualize capsules. The positive stain colors the body of the cell, while the negative stain colors the background but not the capsule

10. Albert Staining Special stains have been developed over time for identifying bacteria species, differentiating them morphologically, and even characterizing there very special features. The most common stain being Gram Staining, Acid-fast staining, endospore staining. Each of these stains aims at identifying and characterizing bacteria based on their morphologies. Albert stain is no different. Its application aim at identifying bacteria that contain special structures known as metachromatic granules. Other staining techniques that are used to detect the presence of granules in the cytoplasmic membrane of bacteria are Nessers’s stain and Pugh’s stain. Albert stain distinctly identifies metachromatic granules that are found in  Corynebacterium diphtheriae .   Corynebacteria are gram-positive, non-spore forming, non-motile bacilli that contain  metachromatic ( Volutin ) granules  which are intracellular inclusion bodies, found in the cytoplasmic membrane of some bacterial cells for storage of complexed inorganic polyphosphate (poly-P) and enzymes. When these granules are subjected to stain with methylene blue dye, they appear reddish-purple color and not the blue dye. The most common Corynebacterium is  Corynebacterium diphtheriae , that caused diphtheria, a nasopharyngeal infection (affecting the nasal, throat) that can also affect the skin, after bacterial colonization and infection. Basically, this bacteria is initially cultured in selective media either a Loeffler agar or Mueller-Miller tellurite agar, or Tinsdale tellurite agar, and its colonize isolated to prepare liquid culture that is then used for staining. Albert stain only acts as a confirmatory stain for the bacteria. Being a differential stain, it can only stain volutin granules, hence bacteria without these granules can not be stained nor identified with this technique .

11. To stain and observe metachromatic granules from a  Corynebacterium diphtheriae  culture . Albert staining technique aims at detecting the presence of metachromatic granulated bodies of  Corynebacterium diphtheriae . Albert stain is made up of two staining solutions; designated as  Albert Solution 1  and  Albert Solution 2,  their compositions being; Albert Solution 1: toluidine blue, malachite green, glacial acetic acid, and alcohol Albert solution 2: Iodine and Potassium iodide in water To use Albert’s staining solutions, each of the two solutions must be prepared effectively with the right percentages of components in order to demonstrate the granules with the right color after staining. Albert staining solution 1 acts as the staining solution while Albert solution 2 acts as the mordant, i.e an ion element that binds and holds a chemical dye, to make it stuck on the micro-organism. Albert Stain 1: Preparation of 100ml Albert stain 1 Into 100ml of water, add 0.1ml of glacial acetic acid Add 2ml of 95% ethanol into the solution Then, dissolve 0.15g of toluidine blue into the solution Lastly, dissolve 0.2g of malachite green into the solution Albert Stain 2: Preparation of 300ml of Albert stain 2 Dissolve 2g of iodine in 50ml of distilled water Add 250ml of water to the solution Dissolve 3g of Potassium Iodide into the solution in the solution Procedure of Albert Staining

12. Albert staining Solution 1 Albert staining solution 2 Distilled water A. Staining: Aseptically, take a loopful culture of  Corneybacterium diphtheriae Make a smear at the center of a clean sterile glass slide Heat fix the smear, gently On a staining rack, place the smeared glass slide. Add Albert staining Solution 1 into the smear and leave it for 3-5 minutes Wash the smeared slide with gently flowing tap water B. Mordanting Add Albert staining solution 2 and leave it for 1 minute Wash the slide with gently flowing tap water. Blot to dry the smeared glass slide Add cedarwood oil on the smear Then observe under a microscope by oil immersion at 1000x Result : The metachromatic granules stain bluish black while the rest of the microbial cell stains green.

13. Interpretation of Albert Staining Corynebacterium diphtheriae  cytoplasmic membrane contains volutin granules, also known as metachromatic granules, which are a characteristic feature of this bacteria. The staining by Albert solutions, stains the granules making the appear as round-shaped blue-black dots at the bottom of L-shaped or V-shaped green Bacilli. Applications of Albert Staining It is majorly used to identify the metachromatic granules found in disease-causing micro-organisms like  Corynebacterium diphtheriae . Being a differential stain, it helps distinguish  Corneybacterium diphtheriae  from other nonpathogenic diphtheroid that lack the metachromatic granules. Limitations of Albert Staining It can only be used to stain the metachromatic granular bodies and not any inclusions in the cytoplasmic membrane.

14. Gram staining Gram staining method, the most important procedure in Microbiology, was developed by Danish physician Hans Christian Gram in 1884. Gram staining is still the cornerstone of bacterial identification and taxonomic division. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition: Gram-positive bacteria  (thick layer of peptidoglycan-90% of cell wall )-  stains purple Gram-negative bacteria  (thin layer of peptidoglycan-10% of cell wall and high lipid content)  – stains red/pink

15. Principle The differences in cell wall composition of Gram-positive and Gram-negative bacteria account for the Gram staining differences. Gram-positive cell wall contains a thick layer of  peptidoglycan  with numerous teichoic acid cross-linking which resists the decolorization . In aqueous solutions, crystal violet dissociates into CV+ and Cl – ions that penetrate through the wall and membrane of both Gram-positive and Gram-negative cells. The CV+ interacts with negatively charged components of bacterial cells, staining the cells purple. When added, iodine (I- or I3-) interacts with CV+ to form large crystal violet-iodine (CV-I) complexes within the cytoplasm and outer layers of the cell. The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of both gram-positive and gram-negative bacteria. The outer membrane of the Gram-negative cell ( lipopolysaccharide layer )  is lost from the cell, leaving the peptidoglycan layer exposed. Gram-negative cells have thin layers of peptidoglycan, one to three layers deep with a slightly different structure than the peptidoglycan of gram-positive cells. With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell. The highly cross-linked and  multi-layered   peptidoglycan  of the gram-positive cell is dehydrated by the addition of ethanol. The multi-layered nature of the peptidoglycan along with the  dehydration  from the ethanol treatment traps the large CV-I complexes within the cell. After decolorization , the gram-positive cell remains purple in color, whereas the gram-negative cell loses the purple color and is only revealed when the counterstain, the positively charged dye  safranin , is added.

16. Classic Gram staining techniques involve the following steps:  Fixation of clinical materials to the surface of the microscope slide either by heating . Application of the primary stain (crystal violet). Crystal violet stains all cells blue/purple Application of mordant: The iodine solution (mordant) is added to form a crystal violet-iodine (CV-I) complex; all cells continue to appear blue. Decolorization step: The decolorization step distinguishes gram-positive from gram-negative cells. The organic solvent such as acetone or ethanol extracts the blue dye complex from the lipid-rich, thin-walled gram-negative bacteria to a greater degree than from the lipid-poor, thick-walled, gram-positive bacteria.  The gram-negative bacteria appear colorless and gram-positive bacteria remain blue. Application of counterstain ( safranin ): The red dye safranin stains the decolorized gram-negative cells red/pink; the gram-positive bacteria remain blue.

17. Procedure of Gram Staining Smear Preparation Fix material on a slide with heat. If the slide is heat fixed, allow it to cool to the touch before applying the stain. Flood air-dried, heat-fixed smear of cells for 1 minute with  crystal violet  staining reagent. Please note that the quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain results. Wash slide in a gentle and indirect stream of tap water for 2 seconds. Flood slide with the mordant:  Gram’s iodine.  Wait 1 minute. Wash slide in a gentle and indirect stream of tap water for 2 seconds. Flood slide with  decolorizing agent (Acetone-alcohol decolorizer ) . Wait 10-15 seconds or add drop by drop to slide until decolorizing agent running from the slide runs clear. Flood slide with a counterstain,  safranin . Wait 30 seconds to 1 minute. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper. Observe the results of the staining procedure under oil immersion (100x) using a Bright field microscope. RESULT:  Gram-negative bacteria will stain pink/red and Gram-positive bacteria will stain blue/purple.

18. INTERPRETATION Gram staining is a microscopic technique that uses a dye to identify bacteria in a sample. The results of a Gram stain can indicate whether there is a bacterial infection, and if so, what type of bacteria it is:  No bacteria: If no bacteria are found in the sample, it's likely that there's no bacterial infection or there weren't enough bacteria to see.  Gram-positive bacteria: Bacteria that appear purple or blue are Gram-positive.  Gram-negative bacteria: Bacteria that appear pink or red are Gram-negative.  Bacteria shape: The most common shapes are rod-shaped (bacilli) or spherical ( cocci ).  Other characteristics: A Gram stain can also indicate the size, quantity, and arrangement of bacteria, as well as whether they are intracellular (present within other cells).  The Gram stain can also detect fungi, which may appear as yeasts or molds.  The Gram stain technique was developed in 1884 by Danish scientist Hans Christian Gram. Gram originally developed the technique to make bacteria more visible in lung tissue, but he later noticed that some bacteria resisted decolorization

19. INTRODUCTION Members of the anaerobic genera  Clostridium  and  Desulfotomaculum  and the aerobic genus  Bacillus  are examples of organisms that have the capacity to exist either as metabolically active vegetative cells or as highly resistant, metabolically inactive cell types called spores. When environmental conditions become unfavorable for continuing vegetative cellular activities, particularly with the exhaustion of a nutritional carbon source, these cells have the capacity to undergo sporogenesis and give rise to a new intracellular structure called the endospore, which is surrounded by impervious layers called spore coats. As conditions continue to worsen, the endospore is released from the degenerating vegetative cell and becomes an independent cell called a free spore. Because of the chemical composition of spore layers, the spore is resistant to the damaging effects of excessive heat, freezing, radiation, desiccation, and chemical agents, as well as to the commonly employed microbiological stains. With  the return of favorable environmental conditions, the free spore may revert to a metabolically active and less resistant vegetative cell through germination. It should be emphasized that sporogenesis and germination are not means of reproduction but merely mechanisms that ensure cell survival under all environmental conditions.

20. PRINCIPLE Malachite Green:  Unlike most vegetative cell types that stain by common procedures, the free spore, because of its impervious coats, will not accept the primary stain easily. For further penetration, the application of heat is required. After the primary stain is applied and the smear is heated, both the vegetative cell and spore will appear green. Decolorizing Agent:  Water Once the spore accepts the malachite green, it cannot be decolorized by tap water, which removes only the excess primary stain. The spore remains green. On the other hand, the stain does not demonstrate a strong affinity for vegetative cell components; the water removes it, and these cells will be colorless. Counterstain ( Safranin ):  This contrasting red stain is used as the second reagent to color the decolorized vegetative cells, which will absorb the counterstain and appear red. The spores retain the green of the primary stain.

21. Procedure of Spore Stain (Schaeffer-Fulton Method) Prepare smears in the usual manner using aseptic technique. Allow smear to air-dry, and heat fix in the usual  manner. Flood smears with malachite green and place on top of a beaker of water sitting on a warm hot plate, allowing the preparation to steam for 2 to 3 minutes.  Note: Do not allow stain to evaporate; replenish stain as needed. Prevent the stain from boiling by adjusting the hot plate temperature. Remove slides from hot plate, cool, and wash under running tap water. Counterstain with safranin for 30 seconds. Wash with tap water. Blot dry with bibulous paper and examine under oil immersion.

22. Endospores: Endospores are bright green. Vegetative Cells:  Vegetative cells are brownish red to pink . Positive: Clostridium perfringens , C. botulinum , C. tetani , Bacillus anthracis , Bacillus cereus, Desulfotomaculum   spp ,  Sporolactobacillus spp , Sporosarcina   spp , etc. Negative: E. coli, Salmonella  spp , etc.

23. Bacterial Cell Wall Staining by Chance's Method Bacterial cell is consisting of various structural components. Cell wall is one of the most important component. Cell wall present outside of the bacterial cell membrane. It gives rigidity, protection and shape to the bacterial cell. Based on the structure of cell wall, all bacteria are divided into two groups as Gram positive and Gram negative. Cell wall of Gram positive cell is monolayered while cell wall of gram negative bacteria is bilayered . Bacterial cell wall can be demonstrated by various special staining methods like Chance's method, Ringer's method & Dyers method. The most common cell wall staining method is Chance's method.

24. PROCEDURE New Fuchsin stain is a basic stain. Therefore it is stained cell wall as well as cytoplasm. Cell wall and cytoplasm both are acidic in nature and they are having negative charge on their surface. However here the strong staining of cell wall take place. Because cell wall is more acidic than cytoplasm due to the presence of free carboxylic groups on its surface . Congo red is a acidic stain and this acidic stain bind with basic stain which is already present on the cell and removes that basic stain. That means here Congo red acts as a decolorizer . but the  removal of basic stain(New Fuchsin ) takes place only from the cytoplasm and not from the cell wall, because it is strongly bound to the cell wall.   Therefore after water wash cytoplasm becomes colourless while cell wall becomes pink coloured . that means here the role of Congo red is to carry out the decolorization , But the decolorization of only cytoplasm takes place and decolorization of cell wall does not take place.

25. Procedure & Steps Prepare the smear on the slide under aseptic conditions with the help of wire loop. Air dry the smear, but do not heat fix (Because the heat fixation changes the structure of capsule. therefore heat fixation is avoided here). Apply 0.5%  New Fuchsin  for 3 minutes. Remove the excess stain (but do not water wash) Apply 0.5 % Congo Red for 4 minutes. Gentle wash with water Air dry and observe under oil immersion objective lens. Spherical and rod shaped bacterial call wall Pink in colour .

26. INTERPRETATION Chance's method for cell wall staining results in an exaggerated width of the cell wall when viewed under a light microscope. This is due to the staining of the cytoplasmic material next to the wall and the formation of a precipitate on the cell surface.  Staining is a technique used to enhance contrast in samples, usually at the microscopic level. Stains and dyes are often used in histology, cytology, and medical fields such as histopathology, hematology, and cytopathology.  Cell walls are structural layers that surround some cell types, and are found outside the cell membrane. They provide the cell with structural support, shape, protection, and help the cell withstand osmotic pressure and mechanical stress.