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Staining of microorganisms (Microbiology).pptx

The document provides a comprehensive overview of bacterial staining techniques, emphasizing the purpose, classification, and methods of staining microorganisms. Key topics include the different types of stains (e.g., acidic, basic, neutral), specific techniques such as Gram staining and acid-fast staining, and their applications in microorganism identification and differentiation. It outlines the mechanisms behind these staining processes and the agents involved, making it a valuable resource for understanding bacteriology.

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1 Staining Jueeli Shiriskar
2 Introduction • Bacteria are very small unicellular microorganisms ubiquitous in nature. • They are micrometers in size. • They have cell walls composed of peptidoglycan and reproduce by binary fission. • Bacteria vary in their morphological features.
3 The most common morphologies are:
4 Staining: According to microbiological definition, stains are dyes or reagents used for differential coloring of microorganisms to observe their structures with much clarity under the microscope. Chemically, a stain/ dye is defined as an organic compound containing a benzene ring plus a chromatographic group.
5 Purpose of staining: To view the organism with much clarity, better in contrast with the background. To determine peculiar structures: there are some special stains that react with certain structures of the organism e.g., spores, cell wall, nuclei, and thus give a clearer picture of such structures. To differentiate one organism from another: some microorganisms under certain conditions take colour and some do not; thus, helps in their differentiation based on staining reactions.
6 Dye or Stain • Dyes are colored organic compound in the form of salt, composed of positive and negative ion, one of these ions is responsible for color called chromogen. • They adhere to a cell and give the color to cell. The presence of color gives the cells significant contrast so they are much more visible. • Different stains have different affinities for different organisms, or different parts of organisms.
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8 Chemically stains are classified as: Acidic dyes: Anionic and stain the cytoplasmic components of the cells which are more alkaline in nature. Eg. Picric acid, acid fuchsin, eosin. Basic dyes: Cationic and combine with those cellular elements which are acidic in nature (nucleic acids) Eg. Methylene blue, crystal violet, safranin. Neutral stains: Formed by mixing an aqueous solution of certain acidic and basic dyes. Eg. Geimsa’s stain, Leishman’s stain, Wright’s stain.
9 Types of stains: Direct or general stains: These stains can stain bacteria directly except for bacterial spores and bacteria with a waxy coating on their cell wall. Indirect stains: These stains stain only the background e.g., nigrosin or India ink. Selective stains: These stains are used to stain parts of the organism like spores, flagella, nuclei, etc. Differential stains: These stains differentiate two groups of bacteria in a mixture viz, Gram-positive and Gram-negative.
10 Staining Techniques Simple Staining Positive Staining Negative Staining Differential Staining Gram Differential Staining Acid Fast Staining
11 Staining techniques:
12 Staining techniques: • 1. Simple staining Technique: • Single staining reagent is used and all cells and their structures stain in the same manner. Positive staining: The stain (methylene blue) is basic (cationic) has a positive charge and attaches to the surface of the object that is negatively charged. Negative staining: The stain (nigrosin) is acidic (anionic) has a negative charge and is repelled by the object (bacteria) that are negatively charged therefore bacterial cells appear transparent. The stain thus stains only the background but not bacteria.
13 Simple staining Technique • Positive staining • Negative staining https://microbenotes.com/negative-staining-principle-procedure-and-result-interpretation/ http://www.cosmosbiomedical.com/catalogues/Microscopy%20&%20Diagnostic%20Staining%20Catalogue% 202016%20WEB.pdf
14 Staining techniques: • 2. Differential staining technique: • More than one staining reagent is used and specific objects (e.g., specific microorganisms or structure of the microorganism) exhibit different staining reactions which are readily distinguishable. • Two widely used differential staining procedures are Gram staining and acid-fast staining.
15 Differential Staining techniques: Acid-fast staining Gram staining
16 Fixation of smear/Fixed Smear Formation: • Fixing before staining it is essential to fix the bacterial sample on to the slide. • The purpose of fixation is to kill the microorganisms, coagulate the protoplasm of the cell and cause it to adhere to the slide. • Heat-fix the specimen on the glass slide, unless the specimen is heat- fixed, the bacterial smear will wash away during the staining procedure. • The staining process involves immersing the sample in dye solution, followed by rinsing and observation. https://www.youtube.com/watch?v=0L97WzZlgXA
17 Mechanism in Simple staining: 1. A stain has a ability to bind a cellular component .These abilities depend upon the charges present on cellular component and charges present on chromophore group of stain. 2. Bacteria has large number of carboxyl group on its surface and these carboxyl group has negative charge. 3. When these carboxyl group carry out ionization reaction it shows COO- and H+ COOH Ionisation COO- + H+ 4. In nature, these H+ ions (unstable) are present on cell surface and further replaced by other positively charged ions like Na+ or K+ and H+ bonds with oxygen to form water.
18 Mechanism in Simple staining: • Basic dyes are available as salts of acids • Eg: Methylene blue chloride (MB.Cl), Malachite green chloride (Mg.Cl) • When these are ionized, MB.CI ionization MB+ + CI- • On addition to methylene blue, exchange of MB+ with Na+ on the bacterial cell take place. • Thus, when colouring agent forms ionic bond with cell or cell components, it results into the staining of cell.
Simple Positive staining 1. With a wire loop place a loop full of bacteria on a clean slide. 2. Place a drop of water over it. Spread the culture so as to form a thin film. 3. Allow slide to dry in the air or by holding it above a bunsen flame. Avoid excess heating. 4. Flood slide with crystal violet and wait for 30 sec. or safranin and wait for 1min. 5. Wash the smear with tap water to remove the excess of stain. 6. Blot dry, then add cedarwood oil and examine under a microscope. 19
20 Simple Positive staining
21 Simple Negative Staining 1. Place a small drop of nigrosin at the end of the slide. 2. Place a loopful of sample and mix with drop of nigrosin. 3. Using the edge of another slide, spread the drop out across the slide. 4. Allow to air dry. 5. Place one drop of immersion oil and examine under a microscope.
22 Simple Negative Staining
23 Gram’s staining Difference between Gram Positive & Gram Negative Bacteria
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25 Difference between Gram Positive & Gram Negative Bacteria
26 Gram’s staining • Gram staining procedure was discovered by Han’s Christian Gram in 1884. • Gram staining is a universal staining technique used for identification and classification of organisms. • In this staining, method bacteria are classified into two groups that are- 1. Gram-positive bacteria 2. Gram-negative bacteria • This classification of bacteria depends upon the property of a cell to retain or lose the primary stain after the treatment of decolorizing agent. • Gram staining is a basic and widely used technique.
27 Requirements: • 1. A clean grease free slide. • 2. Bacterial cell suspension. • 3. Nichrome Wire loop. • 4. Primary stain - Crystal violet. • 5. Mordant- Gram’s Iodine. • 6. Decolorizing agent - 95% alcohol ( 95% Ethanol). • 7. Counterstain- Basic fuschin or Safranin.
28 Procedure: 1. Take a clean grease free slide. 2. Prepare a smear of a bacterial cell suspension on a slide using nichrome wire loop. 3. Air dry and heat fix the smear. 4. Flood the smear with a primary stain (Crystal violet) and allow it to react for 1-2 minutes. 5. After Crystal violet treatment water wash treatment is given to the slide. 6. Further, the smear is treated with the mordant (Gram’s Iodine) for 1-2 minutes. 7. Excess Gram’s Iodine is removed and the slide is further treated with a decolorizing agent (95 % Ethanol). 8. After Ethanol treatment the smear is water washed and flooded with counter stain (Basic fuchsin or Safranin) for 1-2 minutes. 9. Finally, the slide is washed with water, air dried and observed under oil immersion.
29 Gram Staining
30 FUNCTION OF AGENTS USED IN GRAM’S STAINING: • 1. Crystal violet – It is a primary stain and a basic dye it stains all micro- organisms. • 2. Gram’s Iodine – Gram’s Iodine acts as a mordant and it forms a complex with crystal violet that is CV-I complex. This complex increases affinity between cell and stain. • 3. 95% Alcohol (95% Ethanol) – It is a decolorizing agent as well as a lipid solvent. It tries to decolorize the cell by removing the CV-I complex from the cell. • 4. Basic fuschsin or Safranin – It acts as a counter stain. It stains the cells that are decolorized by alcohol. Only Gram-negative bacteria get decolorize and this counter stain gives pink color to these cells.
31 Mechanism of Gram Staining: • 1. When a smear is stained with crystal violet it stains all cells to violet color. • 2. After application of Gram’s Iodine, its molecules acts as a mordant and forms a crystal violet – Gram’s Iodine complex that is CV-I complex. • 3. After CV – I complex formation this smear is subjected to decolorizing treatment by using 95% Ethanol for 30 seconds. • 4. The gram-positive cell has some special features due to which CV – I complex is unable to come outside the cell they are-
32 Mechanism of Gram Staining: • The gram-positive cell has 1 to 4 % of lipid content due to low lipid content the cell get dehydrated by alcohol treatment and its pore size decreases so CV – I complex is trapped inside the cell. • Peptidoglycan layer account about 40 to 90% of the dry weight of Gram-positive cell so due to extremely dense cross-linkage CV – I complex is trapped inside the cell. • The gram-positive cell contains Magnesium ribonucleate so this compound Magnesium ribonuclease molecule forms a covalent bond with CV – I complex and thus it doesn’t allow CV – I complex to come outside the cell. • The gram-negative cell contains 11 to 20 % of lipid content when Gram-negative cells are suspended in alcohol it dissolves the lipid and thus CV – I complex comes out. • Peptidoglycan content in Gram-negative cell wall is 5 to 10 % so due to less amount of cross- linkage CV – I complex comes out easily. • Gram-negative cell lacks Magnesium ribonucleate molecules so CV – I complex is extracted easily from the cell.
33 Mechanism of Gram Staining: • 7. The cells which get decolourised by alcohol take the counterstain and appear pink in color these cells are Gram-negative cells. • 6. After decolourisation treatment, the smear is treated with counterstain i.e Basic fuschin and Safranin. • EXAMPLES • 1. Gram positive bacteria – Bacillus, Staphylococcus, Streptococcus, Micrococcus etc. • 2. Gram negative bacteria – Pseudomonas, E.coli, Salmonella, Shigella, Proteus, Xanthomonas
34 Application of Grams staining: • Gram staining is a basic technique used for identification and classification of the cell • It is a useful technique in the diagnosis of the causative agent of a clinical infection. • It is also helpful in studying morphological characters of cells. • https://www.youtube.com/watch?v=PJmtkQpqJxE
35 Acid Fast Staining: Introduction • In nature, there is a variety of micro-organism and each micro-organism have some special characters. • Most of the microorganisms are easily stained by simple staining procedures. • But there is some micro-organism that is not easily stained by this technique because they have a waxy covering on its surface. If anyhow they get stained they don’t get decolorize even by strong acid. • Such organism requires a special staining technique. • Acid-fast staining technique is a differential staining technique in bacteriology. • This staining technique was discovered by scientist Paul Ehrlich in 1883. • Acid-fast staining technique helps us to differentiate the organism as acid-fast and non- acid fast organisms. • For staining such organism Ziehl- Neelsen staining method is used. It is also called as Acid-fast staining method.
36 Requirements: • 1. A clean grease free slide. • 2. A bacterial cell suspension. • 3. Staining agent- Ziehl Neelsen, carbol fuchsin. • 4. Boiling water bath. • 5. Decolorizing agent – Acid alcohol. • 6. Counterstain – 1% Malachite green or 0.3 % Methylene blue.
37 Procedure: 1. Take a clean grease free slide and prepare a smear using nichrome wire loop. 2. Air dry and heat fix the slide. 3. The slide is flooded with ZNCF stain and placed on a boiling water bath for steaming for about 3-5 minutes. 4. During steaming the stain is repeatedly added on the slide to avoid drying of smear. 5. Further, the slide is treated to the decolorizing agent that is acid alcohol until the stain disappears in washing. 6. After decolourisation, the slide is given a water wash treatment. 7. Further, the smear is flooded with the counterstain that is 1% Malachite green or 0.3% Methylene blue for about 2 minutes. 8. After 2 minutes the slide is washed with water, air dried and observed under oil immersion objective.
38 Procedure:
39 FUNCTION OF REAGENTS USED IN ACID FAST STAINING • 1. ZNCF ( ZIEHL NEELSON OR CARBOL FUCHSIN) • Primary stain. • Many acid-fast bacteria are not stained with the common stain like carbol fuchsin because they are prepared in aqueous solution. • These acid fast bacteria require a stain that is prepared in phenolic stain and ZNCF stain is prepared in phenolic solution. • As these acid-fast bacteria have a waxy covering on their surface and phenolic stain solubilizes waxy covering and stains the cell. • The cells stained with ZNCF appear pink in color.
40 FUNCTION OF REAGENTS USED IN ACID FAST STAINING • 2. ACID ALCOHOL • It is the decolorizing agent. • It is prepared from the combination of acid that is 3% hydrochloric acid and alcohol that is 95% ethanol. • 3. 1% MALACHITE GREEN or 0.3 % MYTHELENE BLUE • It acts as a counter stain. • It stains the decolorized cell and these cells appear green or blue in colour.
41 Mechanism: Acid-fast bacteria have a waxy covering on its surface or high lipid content in the cell wall. The cell wall of acid-fast bacteria is made up of lipids like Mycolic acid and Glycolipids. Due to these high lipid content in the cell wall, these cell wall has less permeability. It is necessary to increase the permeability of the cell wall so the stain can easily penetrate in the cell. The permeability of the cell wall is increased by using phenolic solution and steaming. After the permeability of the cell increases the cells get the stain. Once stained cell doesn’t decolorize even by strong decolorizing agent and appear pink in color. But the non-acid fast bacteria get decolorize easily and get stained by counter stain and appear green or blue in color. If Malachite green is used cells appear green and if Methylene blue is used cells appear blue in color.
42 • SOME IMPORTANT POINTS • 1. The permeability of acid-fast cell is increased by phenolic stain because phenolic stain have high affinity towards the waxy covering and it is more soluble in waxy covering. • 2. For increasing the permeability we use heat steaming because steaming softens the waxy material and allow easy penetration of stain. • APPLICATIONS • 1. Acid-fast staining is useful in the diagnosis of Tuberculosis and leprosy.
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46 Spore Staining • In 1922, Dorner published a method for staining endospores. • Shaeffer and Fulton modified Dorner’s method in 1933 to make the process faster. • The endospore stain is a differential stain which selectively stains bacterial endospores. • The main purpose of endospore staining is to differentiate bacterial spores from other vegetative cells and to differentiate spore formers from non-spore formers.
47 Principle • Bacterial endospores are metabolically inactive, highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions. • The bacteria can remain in this suspended state until conditions become favorable and they can germinate and return to their vegetative state.
48 Principle • In the Schaeffer-Fulton`s method, a primary stain-malachite green is forced into the spore by steaming the bacterial emulsion. Malachite green is water soluble and has a low affinity for cellular material, so vegetative cells may be decolorized with water. Safranin is then applied to counterstain any cells which have been decolorized. At the end of the staining process, vegetative cells will be pink, and endospores will be dark green. • Spores may be located in the middle of the cell, at the end of the cell, or between the end and middle of the cell. Spore shape may also be of diagnostic use. Spores may be spherical or elliptical.
49 Reagents used for Endospore Staining • Primary Stain: Malachite green (0.5% (wt/vol) aqueous solution) 0.5 gm of malachite green 100 ml of distilled water • Decolorizing agent Tap water or Distilled Water • Counter Stain: Safranin Stock solution (2.5% (wt/vol) alcoholic solution) 2.5 gm of safranin 100 ml of 95% ethanol
50 Procedure 1.Take a clean grease free slide and make smear using sterile technique. 2.Air dry and heat fix the organism on a glass slide and cover with a square of blotting paper or toweling cut to fit the slide. 3.Saturate the blotting paper with malachite green stain solution and steam for 5 minutes, keeping the paper moist and adding more dye as required. Alternatively, the slide may be steamed over a container of boiling water. 4.Wash the slide in tap water. 5.Counterstain with 0.5% safranin for 30 seconds. Wash with tap water; blot dry. 6.Examine the slide under microscope for the presence of endospores. Endospores are bright green and vegetative cells are brownish red to pink.
51 Procedure
52 Spore Staining
53 Capsule staining • A bacterial capsule refers to the mucilaginous coating that surrounds the cell wall of bacteria. • The capsule or glycocalyx is composed of glycoproteins. • The cytoplasm of bacteria partially forms a capsule, which then migrates to the cell wall, and there it exists as a mucous or slime covering.
54 Principle • The principle of capsule staining is based on staining of background with an acidic stain and staining of bacterial cell with a basic stain. • A capsule being non-ionic will not stain by either of the two dyes. • Thus, capsule staining creates contrast by staining a bacterial cell along with its background and leaving a capsule as a colourless halo. • The other approach of capsule staining is to stain the capsule by leaving a bacterial cell and background colourless. • Differential capsule stains are available to highlight the specific structure (like capsule) in a bacterial cell.
55 Capsule staining
56 India ink Method • India ink method uses two types of stain, i.e. a basic stain (Crystal violet) and an acidic stain (India ink). • Crystal violet is a positive stain that will stain the negatively charged bacterial cell. • India ink is a negative stain that will stain the positively charged background. • After staining: • The background seems darker or black. • A bacterial cell appears violet. • The capsule seems like a clear halo.
57 Procedure 1.Take a clean, sterilized or grease-free slide. 2.Add a drop of India ink to the centre of the glass slide. 3.Prepare a smear by taking an inoculum from the bacterial culture and mix it with a drop of India ink. 4.Then, allow the smear to air dry (do not heat fix the smear, as it may result in cell shrinkage that in turn can distort the bacterial capsule). 5.Flood a smear with the crystal violet stain and leave it for 30 seconds. Later, remove the extra stain by tilting a glass slide. 6.Add oil immersion to the stained area and observe it under the microscope using a 100X objective.
58 Procedure • India ink method is a type of negative staining method, which stains both the bacterial cell and its background (but not a capsule). • As a result, a capsule appears as a bright halo between the violet bacterial cell and a darker background.
59 Flagella Staining
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