Nuclear Staining by Giemsa’s Method and its Details

Nuclear Staining by Giemsa’s Method and its Details Aim To stain bacteria nuclear material by Giemsa’s staining technique. Approach DNA is a principle genetic material of all micro-organisms except some viruses. Bacterial cell lacks a well-organised nucleus. It is the very essential structure of a living cell. Nucleus performs important functions like growth, metabolic activities, … Read moreNuclear Staining by Giemsa’s Method and its Details

Flagella Staining by Loeffler’s Method and its Details

Flagella Staining by Loeffler’s Method and its Details

Flagella is the most important locomotory organ of a bacterial cell and for studying this structure it is important to stain the flagella.So here we have another staining technique used for staining of flagella that is Loffler’s staining technique.

There are some other staining techniques that are also used for staining of flagella that are Bailey’s method,Gray’s method, Casares-Gil’s method and Leifson’s method.

Here in this article we are going to study  Loffler’s method in detail.

Aim

To stain flagella of a cell by using Loffler’s method.

Requirement

  1. Flagellated cell culture.
  2. Loeffler’s mordant.
  3. Loeffler’s stain.
  4. Distill water.
  5. Chromic acid.

Procedure

  1. The slides are cleaned by boiling them in chromic acid solution.
  2. The smear is prepared as given in the flow chart below.
  3. Further after smear preparation the slide is treated with Loeffler’s mordant and heated it till steam appears approximately it takes 3 minutes.
  4. After that slide is gently washed by distill water.
  5. Further slide is treated with Loeffler’s stain and heated till steam appears.
  6. Finally slide is washed with water,air dried and observe under oil immersion.

Read moreFlagella Staining by Loeffler’s Method and its Details

Spirochete Staining by Becker’s Method

Spirochete Staining by Becker’s Method

  • Spirochete are spiral shape bacteria .Becker’s staining is another special staining used to stain spirochete.
  • Other Spirochete staining techniques are Rue’s method and Levaditi’s method.

Aim

To stain spirochete by using Becker’s method.

Requirement

  1. Tartar sample.
  2. Fontana’s fixative.
  3. Fontana’s mordant.
  4. Becker’s stain.

Procedure

  1. First of all a sterile pin is used to take a sample of tartar from teeth.
  2. The tartar sample is mixed and homogenized in sterile distill water and further used as a suspension.
  3. A drop of suspension is taken on slide ,smear is prepared,air dried and  fixed by using chemical fixative that is Fontana’s fixative for 30 seconds for 2 times after interval of 1 minute.
  4. The slide is further washed with water,air dried and treated with Fontana’s mordant for 5 minutes.
  5. After 5 minutes the mordant is drained off and slide is given a water wash treatment.
  6. Further the slide is treated with Becker’s stain for 3 to 5 minutes.
  7. Finally slide is washed with water,air dried and observe under oil immersion.

Read moreSpirochete Staining by Becker’s Method

Spirochete Staining by Fontana’s Technique

Spirochete Staining by Fontana’s Technique

Introduction

  • Spirochete are spiral shaped Gram negative bacteria.
  • They are unique in morphology and locomotion.
  • The spirochete is spiral in shape so its size is measured by measuring its length and width.
  • Generally, it is 5 to 500 µm in length and 0.1 to 3 µm in width.
  • Spirochete has endo-flagella that runs through periplasm.
  • The endo-flagella maybe 2 to 100 on the number and these flagella are wound around the helical cells in between protoplast and outer membrane.
  • These spirochetes are extremely delicate organism so some special technique is used to stain them.

There are two technique to stain these spiral shape organism.

  1. Fontana’s staining technique.
  2. Becker’s staining technique.

Here we are going to study Fontana’s staining technique in detail.

Fontana’s staining technique

Aim

To stain spirochete by using Fontana’s staining technique.

Requirement

  1. Fontana’s fixative.
  2. Fontana’s mordant.
  3. Fontana’s stain.
  4. 95% Ethanol.
  5. Tartar sample.

Read moreSpirochete Staining by Fontana’s Technique

Rules and Safety Measures in Microbiological Laboratory.

Rules and Safety Measures in Microbiological Laboratory It is very important to follows rules of microbiological laboratory in safety point of view. If these important rules are not followed a student,teacher, or a laboratory worker can face Biohazards, Chemical hazards as well as Physical hazards. The safety rules are as follows Whenever a person enters … Read moreRules and Safety Measures in Microbiological Laboratory.

Flagella Staining Technique by Liefson’s Method

Flagella Staining Technique by Liefson’s Method

Introduction

  • Bacteria have two types of locomotory organs and that are Flagella and pili.
  • Here we are studying about flagella staining.
  • Flagella is a thin,hair like structure made up protein called as flagellin.
  • It size ranges from 20 µ to 200 µ in length.
  • Flagella is one of the most important locomotory organ .It is mainly made up of three parts- 1) Basal body 2) Filament 3) Hook.
  • Flagella is generally present in rod shape bacteria and very few cocci shape bacteria posses flagella.
  • As flagella are very thin and hair like they cannot be easily observed under microscope.
  • So a special technique is design to increase thickness of flagella as well as stain it.
  • Due to these technique we can observe structure of flagella easily under microscope.

Please check out the below video for Flagella staining by Liefson’s method.

Aim

To Stain Flagella of cell by using Leifson’s method

Requirement

  1. Flagellated cell culture slant.
  2. Leifson’s stain.
  3. 1 % Methylene blue.
  4. Distilled water.

Procedure

  • First of all take two hours old flagellated cell culture slant and add two to three drops of sterile distill water in the slant with the help of sterile pipette.
  • Note that the distill water is added slowly without disturbing the growth of cells.
  • After addition of distill water incubated the slant for 20 minutes.
  • Then take a drop of suspension from the slant and place the drop on a clean slide which is kept in slanting position.
  • The drop should flow slowly from one end of slide to other end to avoid folding of flagella on cell.
  • Allow smear to air dry here we don’t use heat fixation treatment .
  • After air drying the slide is flooded with Leifson’s stain till a thin film of shinny surface appear.
  • After this give a gentle stream of water wash treatment to a slide.
  • Now treat the slide with 1 % methylene blue treatment for 1 minute.
  • Give the slide water wash treatment ,air dry and observe under oil immersion lens.

Read moreFlagella Staining Technique by Liefson’s Method

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.

Read moreEndospore staining by Bartholomew and Mittwer’s Method

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.

Read moreEndospore Staining by Dorner’s Method

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.

Read moreVolutin Granule Staining Technique and its Details

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.

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