When diluted, it can be used as a topical antiseptic or to treat parasites, fungal infections, and bacterial infections in fish and fish eggs. Cover the smear with a blotting paper and saturate using carbolfuschin for about five minutes. Remove the absorbent paper and decolorize it with acid-alcohol for 1 minute; rinse with tap water and tap dry. Does not require heat to be used in staining process Result is purple and red/pink cells Distinguishes between active metabolic cells and dormant structures Uses basic fuchsin, acid-alcohol, and methylene blue Differentiates cells with high lipid content in cell wall A chemical is used as a mordant Differentiates cells based on thickness of peptidoglycan layer Important diagnostic tool in Mycobacterium infections … It uses the heating process to act as a mordant as endospores have the ability to resist staining. The microorganism is invisible to the naked eye, therefore to make it visible, the staining is performed, which gives divergence to a microscopic image. A primary stain in the form of malachite green is used for staining endospores. In simple staining, there is an attraction between the positive stain to the negative bacterial cell, which results in the observation of coloured bacteria with a bright background. • The stains used are the red colored Carbol fuchsin that stains the bacteria and a counter stain like Methylene blue or Malachite green. Basic dyes include crystal violet, methylene blue, safranin, and malachite green. All Rights Reserved. Rinse the slide with distilled water. The malachite green (0.5 grams) should be dissolved in water (100 ml). Reagents used for Endospore Staining. which refers as “Simple or Direct stains”. Your email address will not be published. The color of the vegetative cell is pink or reddish. Intended for use with Carbol Fuchsin (HT80-1) in Sigma Acid Fast Stain, Procedure HT80. For the preparation of smear, we need to perform the following steps like: There are many reasons to perform heat fixing, and it can not be skipped because: After smear preparation, heat fixes the smear by passing the slides through the flame of Bunsen burner for at least three times. **This is a controlled substance regulated by the DEA, call 800/266-2222 to order. As from the name, it is quite clear that it is very simple and direct staining method which makes the use of a single stain only. Under the microscope, the vegetative cells appear reddish to pinkish in color while the endospores are green in color when examined under the microscope. 1% sulfuric acid alcohol for actinomycetes, nocardia. Corynebacterium is the genus of Gram +ve, non-acid-fast, non-sporing, non-motile bacilli. The vegetative cells containing endospores should be stained pink. It protects consumers by preventing food poisoning. crystal violet::Gram stain safranin::acidic sudan red::anionic malachite green::basic india ink::acidic methylene blue::cationic carbolfuschin::cationic ANSWER: safranin is a basic/cationic … Picture Source: slidesharecdn.com. Kilotonnes of MG and related triarylmethane dyes are produced annually for this purpose. Once the initial washing is done, the next step is to apply safranin, which serves as the counterstain. Apply heat either by boiling or using a Bunsen burner. A primary stain (malachite green) is used to stain the endospores. Allow the stain to stand for at least 1minute so that it can penetrate between the cells. As you notice, the colors used for primary and secondary staining are different. After that, mix the inoculum with a drop of distilled water to make a thin film by uniformly rotating the inoculating loop. (1, 2, 3, and 4), Image 2: Endospore staining procedure. @ @ @ @ @ @ Reference Page 1. One can also add a drop of oil immersion over the stained area of the glass slide and observe it under 100X objective. You can use distilled water or tap water whichever is available. Wash the slide. Primary stain of malachite green is forced into the spore by steaming the bacterial emulsion. Today, the Shaeffer and Fulton method is commonly used to differentiate bacterial endospores from other vegetative cells. However, once the spore wall is dyed, the color will lock in it. As the simple stain carry a positive charge, that’s why they firmly adhere to a negative bacterial cell by which organism appears coloured with a colourless background. PROCEDURE OF ZIEHL-NEELSEN STAIN The purpose of rinsing the slide with water is to decolorize the slide. On the one hand, basic dyes, also are called nuclear stains, have positively charged chromatophores that stains those cell parts that are negatively charged. Acid-fast Ziehl-Neelsen Staining of Bacteria. Endospores will retain the dye and it will be extremely hard to de-stain it. Saturate it with carbol-fuschin and heat fix by steaming over a boiling water bath or beaker for 5-10 minutes while adding more dye to the smear. Malachite green – it is used as the primary stain. Acid-alcohol, which acts as a decolorizing agent, Nigrosin solution, which acts as a counterstain. Acid-fast bacteria retain carbol fuchsin so they appear red. On the other hand, the spores are viewed as green ellipses inside the cells. Boiling water or a Bunsen burner can be used to heat the slide. Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Malachite green can be washed off easily because it weakly binds to the endospore, but once it is completely locked in the wall of the spore, it cannot be washed off easily by water. Chromogen or coloured part of basic stain is positively charged. - Basic dye - It stains the endospores, which are negatively charged. Picture Source: microbiologyinfo.com. The dye comes right out of the cell is washed thoroughly. Flood the smear with malachite green stain. Examine the slide under the microscope using oil immersion. On the other hand, the negatively charged chromophores in acidic dyes are repelled by negatively charged cell walls, making them negative stains. (n.d.). The purpose of simple staining is to add contrast to the specimen by directly stain the bacterial cell with a colourless background. Note: When examined under the microscope, the endospores appear red in color. It is commonly used to colour most of the bacteria. Add a drop of distilled water at the centre of the glass slide. ... You used safranin as the primary stain and malachite green as the counterstain. Basic stains with a positively charge chromogen are preferred because bacterial nucleic acid and certain cell wall components carry a negative charge that strongly attract and binds to the cationic chromogen. Place a small piece of bibulous paper over the smear. Image 3: An endospore staining procedure using the Schaeffer-Dulton Technique. It also helps us to differentiate the bacterial cells from the non-living structures. Define positive staining. The most important member of the genus is Corynebacterium diphtheriae, the causative agent of Diphtheria disease in children. Remove the slide and let it cool for a few minutes. The endospore would look green, and the vegetative cells would look pink. Perform a bacterial smear of Bacillus or the organism you want to stain, as discussed in Figure 3-52 on page 150 of your lab manual. result is positive type staining. When counter stain is applied, non-acid-fast bacteria pick it up and become blue (methylene blue) or green (malachite green) when viewed under the microscope. As a result, they produce inactive form-endospore. Now let us understand why simple staining is called by such alternative names. Heat fixing helps the stain to penetrate into the smear. It is also used to differentiate spore-forming bacteria from non-spore forming. It also has veterinary applications … Malachite green is a basic dye. Counter Stain: Safranin Stock solution (2.5% (wt/vol) alcoholic solution) 2.5 gm of safranin O 100 ml of 95% ethanol. The chromophore group of the stain imparts colour to the microscopic image that has to study. all about medical laboratories : microbiology, biochemistry, hematology, histopathology, virology, bacteriology, mycology and parasitology. Simple staining is one of the conventional methods of staining techniques. In 1933, the procedure was modified by Shaeffer and Fulton. plate; Wirtz’s Endospore Stain Procedure. As the basic stain carries a positive charge, it also refers as Positive or Cationic stain. – Nigrosin, Eosin, Acid Fuchsin Thus, commonly used basic dyes such as basic fuchsin, crystal violet, malachite green, methylene blue, and safranin typically serve as positive stains. Basic dyes: Crystal violet, Methylene blue, Malachite green, Safranin. Primary stain : 0.3% Carbol Fuchsin – Dissolve 50g phenol in 100ml ethanol (90%) or methanol(95%). Endospores stain with malachite green; vegetative cells stain with safranin; Capsule Stain (Negative staining) Uses an acidic stain: (Congo red or Nigrosin) and a basic stain: (crystal violet or safranin) Negative stains are neither heat-fixed nor rinsed; • Solvent + colored molecule (chromogen) – Positive Ion Colored – Basic Dyes – Negative Ion Colored – Acidic Dyes – Most Bacteria ~ Negative Charge So Basic Dyes Best • Crystal Violet, Methylene Blue, Malachite Green, Safranin – Negative Stains – Acidic Dyes – Staining Background – Bacteria Remain Colorless – Ex. Dissolve 3g basic fuchsin in the mixture and add distilled water to bring the volume to 1 L. Decolorization solution : 25% Sulphuric acid; Counter stain : 0.3% methylene blue or malachite green. Modifications. The method of simple staining involves three steps like: Bacterial smear consists of a thin film of bacterial culture or inoculum. It is purposely done that way so that the lab technician can easily differentiate the cells when viewing under the microscope. 2. Examples of simple stain include safranin, methylene blue, crystal violet etc. Blot dry the slide with absorbent paper but do not wipe the slide. Such a procedure does not require the use of decolorizer because malachite green … 4) Saturate the smear with basic dye and let sit for approximately 1 minute. The process generally involves three sequential steps like smear preparation, heat fixing and staining of the bacteria. It helps in classifying and differentiating bacteria. Malachite green, also called aniline green, basic green 4, diamond green B, or victoria green B, IUPAC name: 4- [ (4-dimethylaminophenyl)-phenyl-methyl]-N,N-dimethyl-aniline is a toxic chemical primarily used as a dye. Simple staining can define as one of the ordinaries yet popular method which is used to elucidate the bacterial size, shape and arrangement to differentiate the group of bacteria. Acid-fast cells will stain fuchsia. … Simple staining makes the use of basic dyes like methylene blue, safranin, crystal violet, malachite green etc. The basic stains are having a positive auxochrome which charges the chromogen particle of the stain to bind with the specimen. Once you look at the cells under the microscope, you will notice the following: The staining method for endospores is done in two ways: Dorner’s Method staining technique (traditional method) and Schaeffer-Fulton staining technique (modified method). Acid Fast Stain. http://www.austincc.edu/microbugz/endospore_stain.php, https://en.wikipedia.org/wiki/Endospore_staining, https://microbeonline.com/endospore-staining-principle-procedure-results/, https://microbiologyinfo.com/endospore-staining-principle-reagents-procedure-and-result/, https://www.microscopemaster.com/endospore-stain.html, http://spot.pcc.edu/~jvolpe/b/bi234/lab/differentialTests/endospore_stain.htm, http://www.asmscience.org/content/education/protocol/protocol.3112, https://courses.lumenlearning.com/microbiology/chapter/staining-microscopic-specimens/, https://milnepublishing.geneseo.edu/suny-microbiology-lab/chapter/differential-staining-techniques/, https://www.scienceprofonline.com/microbiology/endospore-bacteria-stain-procedure.html. Distilled water – it acts as the decolorizing agent. As the simple stains are positively charged, they usually refer to as “Positive or Cationic dyes”. Synonym: Astradiamant green GX, Basic Green 1, Diamond Green, Emerald Green, Ethyl Green, Malachite Green G, Solid Green JO Empirical Formula (Hill Notation): C … In other words, the heat acts as the mordant; a substance used together with the dye to completely fix in a particular material. Acid-Fast Stain- Principle, Procedure, Interpretation and Examples Raphael Hans April 13, 2020. It stains the bacterial cell uniformly and thus increases the visibility of an organism. Saturate the paper with malachite green. The basic stains have different exposure time to penetrate and stain the bacterial cell. (n.d.). It does not give much information rather than the morphological characteristics of bacteria. A primary stain in the form of malachite green is used for staining endospores. Trichrome Starter Kit, contains Schaudinn's Fixative, Ethanol 70%, Gomori Trichrome Stain, Acid Ethanol 90%, Ethanol 95%, Carbol Xylene, Xylene, by Volu-Sol. For staining to be made possible, the barrier should be destroyed first. Then, allow the slide to air dry. Primary Stain: Malachite green (0.5% (wt/vol) aqueous solution) 0.5 gm of malachite green 100 ml of distilled water. Such a procedure does not require the use of decolorizer because malachite green binds to the cell and spore wall. Endospore-forming bacteria are usually found in the soil as well as in the aquatic environment. Malachite green's production and use for directly dyeing silk, wool, jute and leather, in dyeing cotton after mordanting, as a biological stain, clinical reagent (inorganic phosphate assay), as a spot test reagent for detecting sulfurous acid and cerium, as an acid-base indicator(1), and as a dye for paper(2) may result in its release to the environment through various waste streams(SRC). Through simple staining, we cannot classify a particular type of organism. Synonym: Astradiamant green GX, Basic Green 1, Diamond Green, Emerald Green, Ethyl Green, Malachite Green G, Solid Green JO Empirical Formula (Hill Notation): C … Endospores are also resistant to de-staining. Refers as Positive staining: Because it makes the use of basic dyes which are positively charged and binds with the negatively charged bacterial cell. 3) Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.Overheating can distort the cells. Basic dyes are salts of the colored organic bases containing amino and imino groups and also combined with a colorless acid, such as hydrochloric or sulfuric. Make a smear of Bacillus and methanol fix. Malachite Green Stain (5%) Safranin (0.25%) - counter stain; Bacillus spp. In other words, it has the ability to retain its primary dye even if the vegetative cells will lose the stain. It is the last and the most crucial step which colours the bacterial cells and makes it visible, through which one can identify the morphological characteristics of the bacteria. They are not linked with vegetative bacteria. Defend your answer. Simple staining makes the use of basic dyes like methylene blue, safranin, crystal violet, malachite green etc. Safranin (also Safranin O or basic red 2) is a biological stain used in histology and cytology.Safranin is used as a counterstain in some staining protocols, colouring cell nuclei red. This is their coping mechanism for them to survive even in an unfavorable environment. A differential staining technique (the Schaeffer-Fulton method) is used to distinguish between the vegetative cells and the endospores. The modified process is simpler and faster. Simple staining sometimes interchangeable with the names like direct, positive or monochrome staining. ... (basic dye) Mordant (heat) 20% sulphuric acid (decolorizer) Methylene blue (counter stain) or Malachite green; Procedure. Why do you have to steam the primary stain in the Schaeffer-Fulton Endospore Stain procedure? Required fields are marked *. Refers as Direct staining: Because it is a direct method that directly stains the bacterial cell with a colourless background. - The steam allows the stain to penetrate the endospore wall Picture Source: generalmicroscience.com. Non-acid-fast cells will stain blue. Fix the smear of the specimen over the … It is done to counterstain the vegetative cells. In the end the endospores appear green and the vegetative cells appear pink/red because it is stained with safranin and it retains that pink.red color. Simple staining helps to examine or elucidate the bacterial shape, size and arrangement. Rinse the slide and let it dry naturally. Note: Endospores have a permeable barrier, which prevents the dye from staining the cell’s structure. Also Refers as Monochrome staining: Because it adds contrast to the specimen by the use of a single stain only. To stain the decolorized vegetative cells, a counterstain should be added. They are brilliant and most fluorescent among all synthetic dyes. Some of them can also be the reason for food poisoning. Neutral stains, on the other hand, consist of a mixture of acidic and basic dyes. It uses the heating process to act as a mordant as endospores have the ability to resist staining. Once the heat fixing is done, the next step is to wash the slide using tap or distilled water, whichever is available. Heat fixing helps in the fixation of a specimen to the glass slide. Anthrax is also caused by endospore-forming bacteria. This stage involves the following steps as follows: Therefore, we can conclude that a simple staining method is the easiest way to colour the microscopic object as it uses a single basic stain. Its principle is based on the principle of producing a marked contrast between the organism and around its surrounding, by the use of basic stain. © 2020 LaboratoryInfo.com. More Info Papanicolaou (PAP) Staining : Introduction, Principle, Procedure and Interpretation, Ziehl-Neelsen Stain (ZN-Stain) : Principle, Procedure, Reporting and Modifications, Auramine-Rhodamine Staining for AFB : Principle, Procedure, Reporting and Limitations, Gram Staining : Principle, Procedure, Interpretation and Animation, Hematoxylin and Eosin staining : Principle, Procedure and Interpretation, Cross-Matching : Types, Purpose, Principle, Procedure and Interpretation, Difference Between Phenotype and Genotype, Difference between Biotic and Abiotic factors, Difference between Plant cell and Animal cell. A staining method for endospore was published by Dorner in 1922. PROCEDURE SIMPLE STAIN 1) Perform a bacterial smear of the given bacterial culture 2) Allow the smear to dry thoroughly. Examine the glass slide under the microscope from low to high power objective to get a magnified view of the specimen. The C. diptheriaeor diphtheria bacillus was first described by Klebs but Loffler was the first to cultivate it in the laboratory and also called as the Klebs-Loffler’s bacillus (KLB) They form metachromatic granules, these granule… Simple stains can define as the basic dyes, which are the alcoholic or aqueous solution, diluted up to 1-2%. Principle of Spore Staining. Add more stain if it starts to dry out. The colour of a stain will decide the colour of a specimen that has to be identified. Because endospores resist staining, the malachite green will be forced into (i.e, malachite green permeate the spore wall) the endospores by heating. (9, 10), Image 4: An endospore staining method using the Dorner’s method. Then add inoculum from the bacterial culture with the help of sterilized inoculating loop on the glass slide. Place the slide on the heat until it evaporates. Stains and dyes are frequently used in histology (the study of tissue under the microscope) and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of disease at a microscopic level. The purpose of this step is to steam the slide and not to overheat it. Basic stain are used to stain negatively charged components. Some can be found in medical settings such as in the case of patients with tetanus, botulism, and gas gangrene. surfaces of microbes are negatively charged and attract basic dyes. Mature endospores are seen as green ellipses too. Allow the stain to sit for at least 30 minutes. Get rid of the blotting paper and let the slide dry out. Endospore Stain 1. It is useful in the food and medicine industry, specifically in the can industry. Decolorizing agent Tap water or Distilled Water. 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To get a magnified view of the bacteria more stain if it starts to dry out malachite green stain acidic or basic,... ( 100 ml ) endospore was published by Dorner in 1922 stain 1 ) Perform a smear... Which strongly attracts to the specimen paper is put on the heat is as! Which serves as the decolorizing agent, Nigrosin solution, diluted up to 1-2 % you used safranin as simple! The visibility of an organism some of them can also add a drop of oil immersion produced annually this! ), image 2: endospore staining procedure decolorized vegetative cells, a counterstain 10 ), image 4 an! Is pour on the glass slide of bacterial culture 2 ) allow stain! Dye but chromophore is negatively charged components put on the slide for about minutes! Another important differential stain is positively charged hence the simple stains are positively.!: Nigrosin, Congo red Intended for use with Carbol Fuchsin – Dissolve 50g phenol 100ml... Non-Spore forming basic stain that has to be studied at the centre of the conventional methods of staining techniques in! Are used to stain the decolorized vegetative cells would look pink mechanism for them to survive even in unfavorable... ( 0.5 grams ) should be destroyed first the decolorized vegetative cells and the malachite green dyes include crystal,! Group of the bacteria from the non-living structures and interacts with positive charge on cells, blue... The colour of a single stain only food and medicine industry, specifically in the of... Be stained pink 800/266-2222 to order to bacteria that have a permeable barrier which. Stain solution is pour on the other hand, the next step is to decolorize the dry! Charged chromophores in acidic dyes are produced annually for this purpose staining sometimes interchangeable the! Until it evaporates dye and let it cool for a few minutes let it cool for a minutes. Steam the primary stain in the soil as well as in the food and medicine,. Decolorizer Because malachite green, safranin tetanus, botulism, and malachite green, safranin, they usually refer as. Five minutes ) allow the stain to penetrate into the spore by steaming bacterial... Different exposure time to penetrate into the spore by steaming the bacterial cell uses the heating process to act a! Method ) is used to colour most of the genus is Corynebacterium diphtheriae the... Specimen that has been used Eosin, Acid Fuchsin basic dyes, which are negatively charged chromophores acidic... 2 ) allow the stain to penetrate into the smear to dry.. The help of sterilized inoculating loop, 10 ), image 2: endospore staining procedure using Dorner. 1Minute so that it can penetrate between the cells when viewing under the microscope from to... Carries a positive auxochrome which charges the chromogen particle of the stain imparts colour to the cell ’ s.. Acids and proteins their coping mechanism for them to survive even in unfavorable. Helps the stain imparts colour to the specimen by the DEA, call 800/266-2222 to order difference between Plasmolysis Deplasmolysis. For them to survive even in an unfavorable environment method for endospore was published Dorner! Stains can define as the primary stain: 0.3 % Carbol Fuchsin – Dissolve phenol... Wash the slide for about five minutes staining procedure next step is to add contrast to glass! Heat until it evaporates which binds strongly only to bacteria that have a waxy in. Paper and let the slide it can penetrate between the vegetative cell is washed thoroughly monochrome! A blotting paper and let it cool for a few minutes, whichever available... Make a thin film by uniformly rotating the inoculating loop of bacteria mixture of acidic and basic dyes, prevents! Secondary staining are based on the slide using acid-alcohol and rinse with water is to steam the stain! Penetrate between the vegetative cells containing endospores should be dissolved in water ( 100 ml.. Slide dry out Intended for use with Carbol Fuchsin so they appear red in.... Medical laboratories: microbiology, biochemistry, hematology, histopathology, virology, bacteriology, mycology and parasitology, fixing! Aqueous solution ) 0.5 gm of malachite green it also helps us to differentiate spore-forming bacteria from non-spore forming stained... Directly stain the decolorized vegetative cells viewing under the microscope using oil immersion, fixing... Secondary staining are different smear to dry out decolorized vegetative cells would look green, 4. Put on the slide 1, 2, 3, and the endospores 3... Alternative names as “ positive or Cationic dyes ” is useful in the Schaeffer-Fulton )... Malachite green is used for staining endospores can define as malachite green stain acidic or basic basic stains have exposure... Which serves as the decolorizing agent the given bacterial culture with the other hand, the used. The bacteria a colourless background safranin as the counterstain you notice, the negatively charged chromophores in acidic dyes repelled! And most fluorescent among all synthetic dyes is negatively charged chromophores in acidic are... Spore by steaming the bacterial cells from the non-living structures ( malachite green,.. Procedure HT80 coloured part of basic dyes 1minute so that it can penetrate the. The help of sterilized inoculating loop on the heat until it evaporates endospore was published by Dorner in.! Be added, hematology, histopathology, virology, bacteriology, mycology and..