Materials Required: 

1. Clean glass slides2. Inoculating loop

3. Bunsen burner

4. Bibulous paper

5. Microscope

6. Lens paper and lens cleaner

7. Immersion oil

8. Distilled water

9. 18 to 24 hour cultures of organisms

 

Reagents: 

1. Primary Stain         -     Crystal Violet2. Mordant                 -     Grams Iodine

3. Decolourizer          -     Ethyl Alcohol

4. Secondary Stain    -     Safranin

 

 

Procedure: 

Part 1: Preparation of the glass microscopic slide 

Grease or oil free slides are essential for the preparation of microbial smears. Grease or oil from the fingers on the slides is removed by washing the slides with soap and water. Wipe the slides with spirit or alcohol. After cleaning, dry the slides and place them on laboratory towels until ready for use.

 

  Part 2: Labeling of the slides 

Drawing a circle on the underside of the slide using a glassware-marking pen may be helpful to clearly designate the area in which you will prepare the smear.  You may also label the slide with the initials of the name of the organism on the edge of the slide. Care should be taken that the label should not be in contact with the staining reagents.

 

  Part 3: Preparation of the smear 

Bacterial suspensions in broth: With a sterile cooled loop, place a loopful of the broth culture on the slide.  Spread by means of circular motion of the inoculating loop to about one centimeter in diameter. Excessive spreading may result in disruption of cellular arrangement. A satisfactory smear will allow examination of the typical cellular arrangement and isolated cells.

Bacterial plate cultures: With a sterile cooled loop, place a drop of sterile water or saline solution on the slide. Sterilize and cool the loop again and pick up a very small sample of a bacterial colony and gently stir into the drop of water/saline on the slide to create an emulsion.

Swab Samples:  Roll the swab over the cleaned surface of a glass slide.

 

Please note: It is very important to prevent preparing thick, dense smears which contain an excess of the bacterial sample. A very thick smear diminishes the amount of light that can pass through, thus making it difficult to visualize the morphology of single cells. Smears typically require only a small amount of bacterial culture. An effective smear appears as a thin whitish layer or film after heat-fixing.

 

  Part 4: Heat Fixing 

Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and allows the sample to more readily take up stains.

 

Allow the smear to air dry.

After the smear has air-dried, hold the slide at one end and pass the entire slide through the flame of a Bunsen burner two to three times with the smear-side up.

 

Now the smear is ready to be stained.

 

Please Note: Take care to prevent overheating the slide because proteins in the specimen can coagulate causing cellular morphology to appear distorted.

 

Part 5: Gram Stain Procedure

  

1. Place slide with heat fixed smear on staining tray. 

2. Gently flood smear with crystal violet and let stand for 1 minute. 

3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. 

4. Gently flood the smear with Gram’s iodine and let stand for 1 minute. 

5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The smear will appear as a purple circle on the slide. 

6. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-decolorize. 

7. Immediately rinse with water. 

8. Gently flood with safranin to counter-stain and let stand for 45 seconds. 

9. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. 

10. Blot dry the slide with bibulous paper. 

11. View the smear using a light-microscope under oil-immersion.

 

   Differences Encountered in a Real laboratory: 

In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab:

 

1. Always wear gloves, and lab coat.2.  Tie your hair properly to prevent any contamination from the culture you are working

with.

3. After entering the lab, make sure that the microscope is working properly. Oculars and objective lenses should be cleaned before and after each use with lens paper.

4. Adjust the illumination before using the microscope.

5.  Prepare your work space (Laminar Air Flow Cabinet) or lab bench by wiping down the area with disinfectant.

6. Properly adjust the flame of the Bunsen burner. The proper flame is a small blue cone; it is not a large plume, nor is it orange.

7. Wipe the glass slide with spirit and wave the slide over the Bunsen burner to remove any unwanted microorganisms in the slide.

8. Label one side of the glass slide with                                    1. Your initials                    2. The date

9. While flaming the inoculation loop be sure that each segment of metal glows orange/red-hot before you move the next segment into the flame.

10. Once you have flamed your loop, do not lay it down, blow on it, touch it with your fingers, or touch it to any surface other than your inoculums. If you do touch the tip to another surface or blow on it, you will have to re-flame the loop before you proceed to your experiment.

11. Allow your loop to cool before you try to pick up your organism.  If you pick up organism with a hot inoculation loop, your cells will be killed and will affect your results.

12. When removing the caps from tubes, always keep the caps in your hand.  Never set them on the table, as they could pick up contaminants.

13. Always handle open tubes at an angle near to the flame of the burner; never let them point directly up, since airborne or other environmental organisms could fall into the tube and cause contamination.

14. As soon as you transfer the organism into the slide, flame your loop. Never place a contaminated tool on your workbench.

15. Try to prepare a single cell layer of organism (a thin smear). Otherwise the all cells will appear as gram positive in thick area.

16. Do not overwarm the cells. Dry the slide thoroughly prior to heat fixing.

17. Use young, vigorous cultures rather than older cultures for your experiment.

18. Decolorisation step should not exceed the time limit.

19. While washing the slide after staining, do not let the water stream fall directly on the smear. This may disrupt the smear. Let the stream of water flow slowly along the surface, such that only the stain is flooded and the smear is intact.

20. Always prefer to observe under 10X first. This will give you an idea of the location of a good area for observation. After this you may prefer to switch over to 40X.

21. Do not ever observe at a specimen at 100X without oil.

22. While focusing the microscope, glass slides should be handled carefully to avoid the chance of chipping or breaking.

23. After the observation, wipe the microscopic lens with an absorbent paper and cover the microscope properly.

24. Discard all contaminated materials properly and return your supplies to the proper storage locations, and clean up your working area.

25. Always disinfect your work area when you are finished.

26. Ensure proper hand washing before you leave from the laboratory. 

 

Typical Gram-negative bacteria: 

1.  Bordetella pertusis, the causative agent of whooping cough2.  Salmonella typhi, the causative agent of typhoid

3.  Vibrio cholera, the causative agent of cholera

4.  Escherichia coli, the normally benign, ubiquitous, gut-dwelling bacteria

 

Typical Gram-positive bacteria:

 

1. Staphylococci such as Staphylococcus epidermidis and Staphylococcus aureus which is a common cause of boils.

2. Streptococci such as the many species of oral streptococci, Streptococcus pyogenes which causes many a sore throat and scarlet fever and Streptococcus pneumoniae which causes lobar pneumonia.

3. Clostridia such as Clostridium tetani, the causative agent of tetanus (lockjaw).

4. Actinomyces such as Actinomyces odontolyticus which is found in mouth.

5. Species of the genus Bacillus such as Bacillus subtilis which are common microbes living in soil.

 

Generally cocci are Gram-positive but there are exceptions. The most significant from a clinical point of view is the gonococcus, Neisseria gonorrhoea which typically appears as a Gram-negative diplococcus looking very much like a pair of kidney bean.

Objectives: 

1. To differentiate between the two major categories of bacteria: Gram positive and Gram negative.

2. To understand how the Gram stain reaction affects Gram positive and Gram negative bacteria based on the biochemical and structural differences of  their cell walls.

 Click to view the Animation

 

 Principle: 

Staining is an auxiliary technique  used in microscopic techniques used to enhance the clarity of the microscopic image.Stains and dyes are widely used in the scientific field to highlight the structure of the biological  specimens, cells, tissues etc.

 

The most widely used  staining procedure  in microbiology  is the Gram stain, discovered by  the Danish scientist and physician Hans Christian Joachim Gram in 1884. Gram staining is a differential staining technique that differentiates bacteria into  two  groups: gram-positives and gram-negatives. The procedure is based on the ability of microorganisms to retain  color of the stains  used during the  gram stain reaction. Gram-negative bacteria are decolorized by the alcohol,  losing  the  color of  the primary stain, purple. Gram-positive  bacteria  are  not  decolorized by alcohol and will remain as purple. After  decolorization step, a counterstain is used to impart a pink color to the decolorized gram-negative organisms.

 

 

Fig: Hans Christian Joachim Gram

 

Importance of a Gram Stain: 

The Gram stain is a very important preliminary step in the initial characterization  and classification of bacteria.  It is also a key procedure in the identification of bacteria based on staining characteristics, enabling the bacteria to be examined using a light microscope.  The bacteria present in an unstained smear are invisible when viewed using a light microscope. Once stained, the morphology and arrangement of the bacteria may be observed as well. Furthermore, it is also an important step in the screening of  infectious agents in clinical specimens such as direct smears from a patient.

 

The Gram stain procedure enables bacteria to retain color of the stains, based on the differences in the chemical and physical properties of the cell wall.

 

     1. Gram positive bacteria: Stain dark purple due to retaining the primary dye called Crystal Violet in the cell wall.

                           Example: Staphylococcus aureus

 

        Fig: Gram positive bacteria

 

         2. Gram negative bacteria: Stain red or pink due to retaining the counter staining dye called Safranin.

                              Example: Escherichia coli

 

 

          Fig: Gram negative bacteria

 

 Bacterial Morphology: 

Bacteria are very small unicellular microorganisms ubiquitous in nature. They are micrometers (1µm = 10-6 m) in size. They have cell walls composed of peptidoglycan and reproduce by binary fission. Bacteria  vary in their morphological features. 

 

 The most common morphologies are: 

  Coccus (pleural: Cocci):

 

 Spherical bacteria; may occur in pairs (diplococci), in groups of four (tetracocci), in grape-like clusters (Staphylococci), in chains               (Streptococci) or in cubical arrangements of eight or more (sarcinae).

For example: Staphylococcus aureus, Streptococcus pyogenes

 

  Bacillus (pleural: Bacilli):

 

   Rod-shaped bacteria; generally occur singly, but may occasionally be found in pairs (diplo-bacilli) or chains (streptobacilli).

   For example: Bacillus cereus, Clostridium tetani

 

  Spirillum (pleural: Spirilla):

 

     Spiral-shaped bacteria    

For example: Spirillum, Vibrio, Spirochete species.

 

Some bacteria have other shapes such as: 

Coccobacilli: Elongated spherical or ovoid form.

Filamentous: Bacilli that occur in long chains or threads.

Fusiform: Bacilli with tapered ends.

 

 

 

                                 Fig: Different bacterial morphology

 

Gram Stain Mechanism: 

Gram Positive Cell Wall: 

Gram-positive bacteria have a thick mesh-like cell wall which is made up of peptidoglycan (50-90% of cell wall), which stains purple.  Peptidoglycan is mainly a polysaccharide composed of two subunits called N-acetyl glucosamine and N-acetyl muramic acid. As adjacent layers of peptidoglycan are formed, they are cross linked by short chains of peptides by means of a transpeptidase enzyme, resulting in the shape and rigidity of the cell wall. The thick peptidoglycan layer of Gram-positive organisms allows these organisms to retain the crystal violet-iodine complex and stains the cells as  purple.

Lipoteichoic acid (LTA) is another major constituent of the cell wall of Gram-positive bacteria which is embedded in the peptidoglycan layer.   It consists of teichoic acids which are long chains of ribitol phosphate anchored to the lipid bilayer via a glyceride. It acts as regulator of autolytic wall enzymes (muramidases: Bacterial enzymes located in the cell wall that cause disintegration of the cell following injury or death.)

 

 

 

Medical Relevance of Gram Positive Cell Wall:

 

LTA also has antigenic properties that stimulate specific immune responses when it is released from the cell wall after cell death. Cell death is mailnly due to lysis induced by lysozymal activities, cationic peptides from leucocytes, or beta-lactam antibiotics.

 

Gram Negative Cell Wall:

 

Gram-negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall) and lose the crystal violet-iodine complex during decolorization with the alcohol rinse, but retain the counter stain Safranin, thus appearing reddish or pink. They also have an additional outer membrane which contains lipids,  which is separated from the cell wall by means of  periplasmic space.

 

 

Medical Relevance of Gram Negative Cell Wall:

 

 The cell wall of Gram-negative bacteria is often a virulence factor that enables pathogenic bacteria to cause disease. The virulence of Gram-negative bacteria is often associated with certain components of the cell wall, in particular, the lipopolysaccharide ( otherwise known as LPS or endotoxin). In humans, LPS  elicits  an innate immune response characterized by cytokine production and  activation of immune system. Inflammation occurs as a  result of cytokine production, which can also produce host toxicity.

 

Stain Reaction:  

 The four basic steps of the Gram Stain are:

 

 

1) Application of the primary stain Crystal Violet (CV) to a heat-fixed smear of bacterial culture.

 CV dissociates in aqueous solutions into CV+ and  Cl – ions. These two ions then penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ions later interacts with negatively charged bacterial components and stains the bacterial cells purple.

 

 

2) Addition of Gram’s Iodine.

 Iodine (I – or I3 –) acts as a mordant and as a trapping agent. A mordant is a substance that increases the affinity of the cell wall for a stain by  binding to the primary stain, thus forming an insoluble complex which gets trapped in the cell wall.  In the Gram stain reaction, the crystal    violet and iodine form an insoluble complex (CV-I) which serves to turn the smear a dark purple color. At this stage, all cells will turn purple.

 

3) Decolorization with 95% ethyl alcohol.

 Alcohol or acetone dissolves the lipid outer membrane of Gram negative bacteria, thus leaving the peptidoglycan layer exposed and increases the porosity of the cell wall. The CV-I complex is then washed away from the thin peptidoglycan layer, leaving Gram negative bacteria colorless.

 

On the other hand, alcohol has a dehydrating effect on the cell walls of Gram positive bacteria which causes the pores of the cell wall   to shrink. The CV-I complex gets tightly bound into the multi-layered, highly cross-linked Gram positive cell wall thus staining the cells purple.

 

The decolorization step must be performed carefully, otherwise over-decolorization may occur. This step is critical and must be timed correctly otherwise the crystal violet stain will be removed from the Gram-positive cells. If the decolorizing agent is applied on the cell for  too long time , the Gram-positive organisms to appear Gram-negative. Under-decolorization occurs when the alcohol is not left on long enough to wash out the CV-I complex from the Gram-negative cells, resulting in Gram-negative bacteria to appear Gram-positive.

 

4) Counterstain with Safranin 

The decolorized Gram negative cells can  be rendered visible with a suitable counterstain, which is usually positively charged safranin, which stains them pink. Pink colour which adheres to the Gram positive bacteria is masked by the purple of the crystal violet (Basic fuschin is sometimes used instead of safranin in rare situations). 

 

 

Fig: Colour changes that occur at each step in the staining process

 Objective: 

To study and gain expertise on differential and cytological staining techniques.

 

Theory: 

Differential staining is a technique that helps to characterize the microorganisms depending on the difference in the physical and chemical nature of the microorganism. The differential and cytological staining techniques discussed in this chapter help to differentiate between acid fast and non acid fast cells and to visualize the intracellular constituents of the microbial cells including endospores, capsules, metachromatic granules, and flagella. In this technique the differential stains applied on the bacterial smear reveals the different types of cells at one point, reveals one part of the cell with one color and other parts a different color.

 

Differential Staining

Gram stain technique is a differential staining technique, which separates bacteria into two groups (discussed in earlier chapters), Gram-positive bacteria and Gram-negative bacteria. Another differential staining technique is acid-fast technique which differentiates species of Mycobacterium from other bacteria. Mycobacterium species due to its special cell wall resist the effect of the decolorizer acid-alcohol and retain the color of the primary stain carbolfuchsin stain and stains the acid fast cells in bright red color. Other bacteria lose the stain and take on the subsequent color of the counter stain methylene blue stain and stain the cell as blue. Endospore staining is a special staining technique, to observe bacterial spores, where the spores take the color of the primary stain Malachite green, while the counterstain, safranin, give color to the non-spore forming bacteria. Specific stains such as nigrosine, Indian ink etc help to visualize the bacterial stains which cannot be stained by usual staining methods. The metachromatic granules, the characteristic feature of Cornybacterium diphtheriae, can be differentiated from the bacterial cells with the help of Albert staining techniques. Flagella are the thin delicate structure for bacterial motility. The thin structures of the bacterial flagella make it difficult to observe under bright field microscope. Special stains

and mordents such as Leifson’s stain are required for staining the bacterial flagella. 

 

Endospore Stain: 

Schaeffer-Fulton Method

 

Endospore staining is used to visualize specialized cell structures. The endospore stain is used to determine the highly resistant spores of certain microorganisms within their vegetative cells. The multiple thick coats of the spore made the endospore resistant to stain with most dyes. In Schaeffer-Fulton method, the primary stain, Malachite Green, is added over the heat fixed bacterial smear and heated over a steam bath for few minutes. This will soften the hard outer coverings of the spore and the primary stain gets stick to the spore. When taken from the steam bath followed by further cooling hardens the outer layer of the spore. During this stage both the spore and vegetative cells appear as green in color. But later the thick outer layer makes the spore resistant to the action of decolorizing agent (water), but however, water can easily decolorize the vegetative cells.  When counterstained with Safranin, vegetative cells are easily stained with Safranin, and the cells appear in red or pink color.

 

Dorner Method

 

Dorner method is an alternative method for staining the endospores. In this staining process, 2- 3ml broth culture of microorganism and equal volume of  Basic fuchsin is heated in a water bath at 100o C for 10 minutes. The extensive boiling softens the structure of the bacterial spores and the basic fuschin get into the spores. After the boiling process, the microbial culture-basic fuschin stain is allowed to cool for sometime which hardens the outer covering of the spore. In order to give a color to the background and to differentiate between the vegetative cells and endospores, a thin film of loopful of the microbial culture-basic fuschin stain and 2 nigrosin on a clean glass slide. Since nigrosin is negative charged, the bacterial cells cannot easily taken up by the cells. After staining the vegetative cells appear become colorless, the endospores stains as red which can be present as terminal or sub terminal. The background is stained as dark by the Negrosin stain.

 

Acid Fast Stain: 

Ziehl Neelsen Method

 

The Ziehl Neelsen Method is used for staining the Mycobacterium in clinical specimens. The thick outer waxy covering (mycolic acid) of the Mycobacterium cell walls act as a barrier  and does not allow all the stains to enter into the cell. In order to visualize these cells higher concentrations of the staining solution is needed and once this stain enters the cell, it is too difficult to remove the stain using a decolorizer. When the clinical specimen is stained with basic dyes such as carbolfuchsin (primary stain) with the continuous application of heat, softens the waxy lipid outer covering of the cell wall and the stain readily enters the cell and stains the cell cytoplasm. When decolorizing agents such as acid-alcohol is added over the primary stain, some bacterial cells cannot be easily decolorized and such bacterial cells are called as acid fast bacteria. The bacteria with high concentration of lipid are easily decolorized by the decolorizing agent and are said to be non-acid fast bacteria. Finally, the addition of the counter stain, Methylene blue, dyes the colorless non acid fast cells as blue thus differentiating them from the pink acid fast bacteria which are unaffected by the Methylene blue.

 

Capsule Stain: 

Capsules are the gelatinous outer layer of the bacterial cells and these structures cannot retain the color of the staining agents. The capsules can be visualized by means of two methods.

 

 Positive Capsule Staining

 

Since capsule is water soluble in nature, it is too difficult to stain the capsule with normal staining methods. The positive capsule staining method (Anthony Method) uses two reagents to stain the capsular material. The primary stain Crystal violet is applied over a non heat fixed bacterial smear so that both the bacterial cells and capsular material take up the color of the primary stain. The ionic nature of the bacterial cell binds the crystal violet stain more strongly while the non-ionic nature of the capsule get adhere with the crystal violet stain. When the decolorizing agent copper sulfate is added over the bacterial smear, the loosely adhered crystal violet stain is washed off from the capsular material without removing the tightly bound crystal violet from the cell wall. The capsular material absorbs the light blue color of the copper sulfate in contrast to the purple bacterial cell.

 

Negative Capsule Staining

 

Another simple method to visualize the bacterial capsules is by using negative staining Technique. During staining the non heat fixed bacterial smear with the acidic stains such as Nigrosin will not penetrate the bacterial cells (since both acidic stain and bacterial surface has negative charge). Instead the acidic stain deposits around the bacterial cells and create a dark back ground and the bacteria appear as unstained with a clear area around them, capsule.

Note: If you heat fix the bacterial smear for capsule staining, the cells will shrink creating a hallow zone around the bacterial cell and will be mistaken for the capsule.

 

Metachromatic Granule Staining: Albert’s Staining

 Albert’s staining is specially demonstrates the presence or absence of the metachromatic granules, a characteristic feature of Cornybacterium diphtheriae. During gram staining if a smear appears as purple rods with straight or slightly curved with clubs at the end, with a characteristic V shape then it is suspected as Cornybacterium diphtheriae. The further confirmation can be done by Albert’s staining technique. This techniques employ two stains Albert Stain A( combination of Toluidine blue, Malachite green, Glacial acetic acid, alcohol and distilled water) and Albert Stain B(Iodine, potassium iodide and distilled water). After the staining process the metachromatic granules appear as bluish black where as the cell appears as green color.

 

Flagella Staining

 Bacteria are motile by means of flagella. The flagella are too thin to be seen in ordinary stains, special stains and techniques needed to visualize the flagellum enough stain to obtain a visible thickness. Specialized stains are usually found in microbiology laboratories to detect the presence or absence of flagella to indicate the nature of that bacterium (motile/non-motile).  

 

Liefson Stain

 

When a bacterial culture is stained with Liefson stain, the tannic acid component of the stain produce a colloidal precipitate which can be taken up by the bacterial flagella will become colorized which can be easily visualized using microscopy. The concentration of the tannic acid and dye is important in staining the bacterial flagella while the alcohol concentration in the Liefsons stains helps in maintaining the solubility of the components. On microscopic observation, the bacterial cells and flagella will stain red and the flagellar arrangement can be visualized easily. The age of the bacterial culture, condition of staining solutions, concentrations of the staining solution etc can also affect the staining reaction.

Materials Required: 

Endospore Staining

 

Schaeffer-Fulton Method

1. Clean glass slide in box

2. Inoculation loop

3. Test organism

4. Absorbent paper

5. Boiling water in tripod stand

6. Water in wash bottle

7. Bibulous paper

8. Tweezers

9. Microscope

10. Primary Stain – Malachite green

11. Counter stain - Safranin 

Staining Steps:

 

1. Aseptically transfer the bacterium with an inoculating loop to a clean glass slide and prepare a thin smear of the bacterium.

2. Air dry and heat-fix the bacterial smear.

3. Cover the bacterial smear with a piece of absorbent paper cut to fit the smear and slide.

4. Place the slide over a container of boiling water.

5. Saturate the absorbent paper with malachite green stain solution and steam for 5 minutes.  Keep the paper moist by adding more stain as required.

6. Remove the absorbent paper using forceps, allow the slide to cool, and rinse the slide with water for 30 seconds.

7. Wash the slide with water.

8. Counter stain with safranin for 30 seconds.

9. Wash the slide with water and blot dry the slide.

10. Examine the slide under the oil immersion lens for the presence of endospores.   

  Expected Result:

 

 On microscopic observation endospores appear in green color and the vegetatives cells as pink.

 

 

Dorner Method

 

1. Test organism2. Glass slides in the box

3. Inoculation loop

4. Basic fuchsin

5. Negrosin

6. Microscope

7. Water bath

 

Staining Steps:

 

1. Mix an aqueous suspension of bacteria with an equal volume of basic fuchsin in a test tube.

2. Keep the test tube in a boiling water bath for 10 minutes.

3. Add a loopful of the boiled basic fuchsin-organism suspension to one side of the glass slide.

4. 7% nigrosin stain is added over the boiled basic fuchsin-organism suspension.

5. Make a thin smear of the culture using a second glass slide making a 45 degree angle with the first slide.

6. Air dries the smear for some time.

7. Examine the slide under the oil immersion lens for the presence of endospores.

 

  Expected Result:

 

On microscopic observation, endospores will appeared as red, and the vegetative cells as colourless, in a dark background.

 

Acid Fast Staining 

Ziehl Neelsen Method

 

1. Clean glass slide in the box2. Inoculation loop

3. Test organism

4. Absorbent paper

5. Beaker with water in tripod stand

6. Tweezers

7. Bibulous paper

8. Microscope

9. Primary stain - Carbol fuschin

10. Decolorizer    - 25% sulphuric acid

11. Counter stain - Methylene blue 

 

Staining Steps:

 

1. Aseptically transfer the bacterium with an inoculating loop to a clean glass slide and prepare a thin smear of the bacterium.

2. Heat- fixes the bacterial smear.

3. Cover the bacterial smear with a piece of absorbent paper cut to fit the smear and slide.

4. Place the slide over a container of boiling water.

5. Saturate the paper with carbol fuschin steam for 5 minutes.  Keep the paper moist by adding more stain as required.

6. Remove the absorbent paper using tweezers, the excess stain is washed with water and allow the slide to cool for some time.

7. Decolorize the slide with 25 % sulfuric acid.

8. Rinse the slide with water.

9. Counter stain the cells with Methylene blue stain for 45 seconds.

10. Wash the slide with water and blot dry the slide.

11. Examine the slide under the oil immersion lens to observe the acid fast or non acid fast-cells

 

Expected Result:

 

On microscopic observation, the acid fast bacterium will appear as pink coloured cells.

 

Capsule Staining

 

Positive Capsule Staining

 

1. Glass slides in the box2. Inoculation loop

3. Test organism

4. Crystal violet

5. 20% copper sulfate

6. Bibulous paper

7. Microscope

 

Staining Steps:

 

1. 2 drops of Crystal Violet (primary stain) is added to one side of the glass slide.2. A loopful of bacterial culture is mixed with Crystal Violet stain.

3. Make a thin smear of the culture using a second glass slide making a 45 degree angle with the first slide.

4. Decolorize and counter stain the bacterial smear with 20% copper sulfate.

5. Air dries the smear for some time.

6. Examine the slide under the oil immersion lens for the presence of capsules of bacteria. 

Negative Capsule Staining 

1. Glass slides in the box

2. Inoculation loop

3. Bunsen burner

4. Test organism

5. Negrosine

6. Microscope

 

Staining Steps:

 

1. Add one drop of nigrosin onto the end of a clean slide.2. A loopful of bacterial culture is added onto the drop of nigrosin and mix well.

3. Make a thin smear of the culture using a second glass slide making a 45 degree angle with the first slide.

4. Air dries the smear for some time.

5. Examine the slide under the oil immersion lens for the presence of capsules of bacteria.

 

  Expected Result:

 

On microscopic observation, the capsulated bacterium can be observed in a dark background.

 

Metachromatic Granule Staining

 

Albert’s Staining

1. Glass slides in the box

2. Inoculation loop

3. Test organism

4. Albert stain A

5. Albert stain B

6. Water in the wash bottle

7. Bibulous paper

8. Microscope. 

Staining Steps:

 

1. Aseptically transfer the bacterium with an inoculating loop to a clean glass slide and prepare a thin smear of the bacterium.

2. Air dries the smear for some time.

3. Cover the air dried smear with Albert Stain A.

4. Remove the excess stain by washing with water.

5. Cover the bacterial smear with Albert Stain B. Let it stand for 2 minutes.

6. Wash the slide with water and blot dry the slide.

7. Examine the slide under the oil immersion lens for observing the metachromatic granules of bacteria. 

Expected Result:

 

The metachromatic granules appear as bluish black and the bodies of the bacillus appear as green.

 

Flagella Staining

 

Liefsons Flagella Stain

1. Clean glass slides in the box

2. Inoculation loop

3. Test organism

4. Leifson’s stain

5. Water

6. Microscope

 

Staining Steps:

 

1.  Make a rectangular border on the surface of the glass slide with a grease pencil.2. Aseptically transfer the bacterium with an inoculating loop to a clean glass slide.

3. Allow the smear to run down in an inclined slide.

4. Flood the slide with Liefson's Flagella Stain and allow staining for 10 – 15 minutes. A fine rust colored precipitate forms throughout the slide.

5. Air dries the slide.

6. Examine the slide under the oil immersion lens to observe the type of flagella. 

Gray Method

1. Nutrient agar plate with test organism

2. Scalpel

3. Tweezers

4. Glass slides in the box

5. Basic fuschin

6. Microscope  

 

Staining Steps:

 

1. Cut a small portion of the agar with the test organism using a sterile scalpel.2. Raise the agar block with the aid of a forceps.

3. Place the agar block in a sterile glass slide, with the bacterial culture facing downwards. Keep it undisturbed for some time.

4. Take the agar block and discard it in hazardous box.

5. Allow the bacterial culture in the glass slide to air dry for some time.

6. Flood the bacterial culture with basic fuchsin until a golden precipitate is observed.

7. Gently wash with water.

8. Examine the slide under the oil immersion lens to observe the type of flagella. 

 Expected Result:

 

On microscopic observation the flagella present around the bacterial cells are observed.