Endospore staining by Bartholomew and Mittwer’s Method

Endospore staining by Bartholomew and Mittwer’s Method

Aim

To stain Endospore of a bacterial cell by Bartholomew and Mittwer’s Method.

Requirement

  1. Cell suspension of endospore producing bacteria.
  2. Malachite green stain.
  3. Saffranin stain.

Procedure

  1. Take a clean grease free slide and prepare a thick smear on a slide.
  2. The smear is heat fixed by passing the slide from the flame for about 25 times.
  3. The slide is allowed to cool.
  4. Further the slide is treated with Malachite green stain and allowed it to react for about 10 minutes.
  5. After 10 minutes slide is given a water wash treatment.
  6. Further the slide is treated with counter stain that is saffranin for about 30 seconds.
  7. After 30 seconds the slide is water washed, air dried and observed under oil immersion.

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Endospore Staining by Dorner’s Method

Endospore Staining by Dorner’s Method

Introduction

  • Endospore is a resistant refractile structure formed inside the cell.
  • Endospore is generally formed by some gram positive bacteria.
  • When a bacteria come across some unfavorable conditions it gives rise to resistant structure called as endospore.
  • These endospore is metabolically inactive,it has low moisture content and it is made up of several layers.
  • Endospore of bacterial is different from fungal spores.
  • It is resistant to heat, freezing, desiccation, radiation and chemical agents.
  • So staining of such a resistant structure is not a easy job.
  • The endospore structure doesn’t get stain easily by a simple staining procedure it requires a technique.
  • Here we have two staining techniques and they are
  1. Dorner’s staining technique.
  2. Bartholomew and Mittwer’s staining technique.

Dorner’s method

Aim

To stain endospore of bacterial cell by Dorner’s method.

Requirement

  1. Cell suspension of sporulating bacteria.
  2. ZNCF stain.
  3. 10 % Nigrosine stain.

Procedure

  1. Take a thick cell suspension and add two drops of distill water.
  2. Further add equal amount of ZNCF stain and keep this tube containing mixture in boiling water bath for 15 minutes.
  3. After 15 minutes take a loopful of suspension from the tube and make a smear on a clean grease free slide.
  4. Air dry and heat fix the slide.
  5. Further take a drop of nigrosine on one end of a slide and make a thin film of a stain all over the smear with the help of other slide.
  6. Allow the film of  Nigrosin to air dry.
  7. After air drying observe the slide under oil immersion.

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Volutin Granule Staining Technique and its Details

Volutin Granule Staining Technique and its Details

Introduction

  • Micro-organism have a special characteristic these organism store reserve food material in the form of granules.These granules are utilized by these organism when they are in starvation mode.
  • These reserve food granules are also called Volutin granules, Metachromatic granules or Babes Ernst granules.
  • These granules are mainly of four types.
  1. Volutin granules.
  2. Sulphur granules.
  3. Nitrogenous reserve food material.
  4. Non-nitrogenous reserve food material.
  • These granules are called as Volutin granules because these granules were first observed in Spirillum volutans.
  • It is also called as metachromatic granule because if these granules are stain with blue stain they appear red in colour so it has a property of changing colour.
  • These granules are made up of proteins, RNA and poly-phosphate molecules .
  • Volutin granules are mainly present in bacteria, algae,fungi and protozoa.
  • These granules are stain by using Albert’s staining method.

Aim

To stain volutin granules by Albert’s staining method.

Requirement

  1. Cell suspension.
  2. Albert’s stain.
  3. Albert’s iodine.

Procedure

  1. Prepare a smear on clean grease free slide.
  2. Air dry and heat fix the smear.
  3. Treat the smear with Albert’s stain and allow it to react for about 3 mins .
  4. Drain of the excess stain do not water wash the slide.
  5. Flood the smear with Albert’s iodine for 2 minutes.
  6. Wash the slide with water,air dry and observe under oil immersion lens.

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Capsule Staining Technique by Maneval’s Method.

Capsule Staining Technique by Maneval’s Method.

Maneval’s staining method is a negative staining method.

Aim

To stain a  capsule of a cell by Maneval’s staining method.

Requirement

  1. 1 % Congo red solution.
  2. Maneval’s stain.
  3. Capsulated cell culture.

Please watch the Video given Below

Procedure

  1. Take a loopful of capsulated cell suspension on a clean grease free slide.
  2. Add a drop of 1 % congo red solution in the suspension and then spread it gently on the slide to form a smear.
  3. Allow the suspension to air dry and do not heat fix it.
  4. Flood the slid with Maneval’s stain and keep it for 2 minutes.
  5. After 2 minutes discard excess stain and do not water wash the slide.
  6. Allow the slide to air dry and observe under oil immersion objective.

Read moreCapsule Staining Technique by Maneval’s Method.

Capsule Staining Technique by Hiss method

Capsule Staining Technique by Hiss method

Introduction

  • Capsule is the rigid, slimy and gummy covering of the bacterial cell which lies external to the cell wall.
  • The cell that posses the capsule are called as capsulated cell and those who lack the capsule are called as the non-capsulated bacteria.
  • Capsule is mainly made up of organic compound like Polysaccharides or polypeptides.
Capsule is mainly of two types
  1. Micro-capsule – Its size is less than 0.2 µ.
  2. Macro-capsule – Its size is more than 0.2 µ.
  • When we stain the cell with normal stain like methylene blue or saffranin it stains the cell as well as capsule as capsule get stained easily.
  • So if we want to stain a capsule we need a special capsule staining technique.
Capsule staining can be done in two ways they are
  1. Positive staining method – In positive staining method only capsule is stained.
  2. Negative staining method – Here capsule is not stained but it is made visible by colourless area of capsule in between coloured cells and coloured background.

So here we are going to study the capsule staining technique and its details.

The two methods which we are going to study are…

  1. Positive staining method – Hiss Staining method.
  2. Negative staining method – Maneval’s method.

Read moreCapsule Staining Technique by Hiss method

Cell wall staining by Chances method

Cell wall staining by Chances method

Please watch the video given below

Introduction

  • Cell wall staining technique is a special staining technique in which we especially stain the cell wall of the bacterial cell.
  • As we know cell wall is the outer most rigid covering of cell and depending upon the structure of cell wall bacterial cell are classified in Gram positive and Gram negative cells.
  • Staining of bacterial cell wall helps us in a demonstration of the cell wall.
  • There are various staining techniques used to stain cell wall like Chances method, Ringer’s method and Dyar’s method.

Aim

  • To stain the bacterial cell wall by Chances method.

Requirement

  1. A Clean grease free slide.
  2. Fresh cell suspension.
  3. 0.5  %  New fuchsin solution.
  4. 0.5  %  Congo red solution.

Procedure

  • A clean grease free slide is taken.
  • The smear is made on a slide by using a sterile wire loop.
  • The slide is air dried and not heat fixed.
  • After air-drying the slide is flooded with 0.5  %  New fuchsin solution and allowed to react for 3 mins.
  • After 3 mins excess stain is drained out and the slide is flooded with 0.5  %  Congo red solution and kept for 4 mins.
  • Further, the slide is gently washed with water.
  • Air dried and observed under oil immersion lens.

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Acid Fast Staining Technique and its Details

Acid Fast Staining Technique and its Details

Introduction

  • In nature, there is a variety of micro-organism 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 are don’t get decolourise even by strong acid.
  • Such organism require 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.

Definition

  1. Acid-fast organism- The organism that get stained by acid-fast staining technique but don’t get decolourised even by strong acid are called as an acid-fast organism.
  2. Non-acid-fast organism- The organism that easily gets stained by a staining procedure as well as decolourises easily by a strong acid are a non-acid fast organism.

Requirement

  1. A clean grease free slide.
  2. A bacterial cell suspension.
  3. Staining agent- Ziehl Neelsen carbol fuchsin.
  4. Boiling water bath.
  5. Decolourising agent – Acid alcohol.
  6. Counter stain – 1% Malachite green or 0.3 % Methylene blue.

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Gram Staining Procedure and its Details

Gram Staining Procedure and its Details

Please watch the video given below

Introduction

  • 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 decolourising agent. 
  • Gram staining is a basic and widely used technique.
  • This technique was modified by many scientists but the best result was obtained by Hucker and Conn’s modification.

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. Decolourising agent- 95% alcohol ( 95% Ethanol).
  7. Counterstain- Basic fuschin or Safranin.

Read moreGram Staining Procedure and its Details

Negative Staining Method and its Mechanism

Negative Staining Method and its Mechanism

Introduction

  • Negative staining procedure is also called as Relief staining.
  • Some bacteria have non-ionic charge or slime layer on their surface.
  • Because of which the stain doesn’t penetrate inside the cell and the bacterial cells are not easily stained.
  • In case of such bacterial cells negative method is used for observing the morphological characters of the cell.
  • In negative staining method actually, the background is stained and the bacterial cell remains colourless.
  •  This is the only staining method in which bacterial cells are not stained but they are made visible against a dark background.
  • Generally, the acidic stain such as 10 % Nigrosin, Eosin, India ink and Congored is used.

Procedure

  1. A grease-free slide is taken and a smear is made on the slide by using Nichrome wire loop.
  2. The slide is allowed to air dry.
  3. Here there is no Heat fixation step.
  4. After air drying a drop of stain is added on an end of a slide and a thin film of stain is made by using another slide.
  5. These film is allowed to air dry and observed under oil immersion.

Read moreNegative Staining Method and its Mechanism

Simple Staining Procedure and its Mechanism

Simple Staining Procedure and its Mechanism

Introduction

  • Simple staining is a method of staining in which bacteria are stained by using a single stain.
  • Simple staining is also called as monochrome staining or positive staining.
  • Examples of simple stain are Methylene blue, Safranin, Malachite green, Basic fuchsin and crystal violet etc.
  • In simple staining procedure cell are uniformly stained.

Please check out the below given video on Simple Staining procedure and mechanism

 

Procedure of simple staining

  1. A clean grease free slide is taken .A grease free slide is is made by first washing the slide with detergent wiping the excess water and the slide is passed through flame.
  2. On these grease free slide smear is made by using a sterile wireloop and cell suspension.
  3. These slide is allowed to air dry .
  4. After air drying these slide is rapidly passed through a flame for three to four times for heat fixation.
  5. After heat fixation the slide is placed on the staining rack and flooded with a particular stain and these stain is allowed to react for three minutes.
  6. Futher the slide is washed under running water.
  7. The slide is air dried and washed under oil immersion.

Read moreSimple Staining Procedure and its Mechanism

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