Aim: Microbial Staining Methods

BACKGROUND

The most of bacteria have refractive indices that are similar to those of the aqueous fluids in which they are floating and are mostly transparent and colourless. For visualization of the bacteria, we use stain and dyes.

To improve the clarity of the microscopic image, staining is an adjunct technique used in microscopic techniques. In the scientific community, stains and dyes are frequently used to emphasize the structure of biological objects, cells, tissues, etc.

Due to their tiny size, bacteria cannot be observed in great detail under a standard light microscope unless they are dyed. Due to their varied staining characteristics for particular bacteria, some staining techniques have significant diagnostic significance. They help to reveal their internal structure.

Simple Staining

The act of colouring a bacterial organism only with one stain is known as simple staining.

Requirement

1. Clean Glass slide
2. Inoculation loop
3. Bunsen burner
4. Microscope
5. Immersion oil
6. 24 hour grown culture of organism
7. Crystal violet or Carbol fuschin.

Procedure

1. Take a clean glass slide, add loopful of the culture of organism.
2. Allow it to air dry and heat fix the glass slide.
3. Flood smear with Crystal violet or Carbol fuschin, wait for 30 to 60 sec.¹
4. Wash the slide under running tap water, to remove excess stain.²
5. Allow to air dry, then add immersion oil and observe under a microscope (100x).

Conclusion

The culture organism observes in purple colour under the microscope.

Negative staining

Negative staining is also known as background staining. The stains in this method are not penetrate the microorganism, in its place, they remove the background, making the organism clear and visible in a dark light field.

Requirement

1. Clean Glass slide
2. Inoculation loop
3. Bunsen burner
4. Microscope
5. Immersion oil
6. 24 hour grown culture of organism
7. Nigrosine or India ink.

Procedure

1. A clean glass slide should have a small drop of nigrosine at the end of it.
2. Take a loopful of culture sample and mix it with the drop of stain.²
3. Utilizing the edge of another slide, spread the drop out over the slide.¹
4. Allow to air dry.
5. Add drop of immersion oil and observe under microscope (100x).

Conclusion

The culture organism observes without any colour, with dark background.

Hanging Drop Technique

Hanging drop technique used to check motility of organisms, therefore it also called as motility test. This technique used to observe living bacteria under microscope.

Requirement

1. Clean cavity Glass slide
2. Cover slip
3. Inoculation loop
4. Bunsen burner
5. Microscope
6. 24 hour grown culture of organism
7. Paraffin wax

Procedure

1. Apply paraffin wax on the edges of the clean cover slip.²
2. Take loopful of culture sample and place a drop in the centre of cover slip.
3. Place cover slip inverted position over the cavity of glass slide.¹
4. Observe under the low power objective (40x) along with the low light.

Conclusion

The motile organism observes the microscope successfully.

Gram’s Staining

Developed by Christian Gram in 1884, this method divides bacteria into two groups based on how they react to the stain: those that retain it are known as Gram-positive bacteria, while those that lose their colour are known as Gram-negative bacteria.

Requirement

1. Clean Glass slide
2. Inoculation loop
3. Bunsen burner
4. Microscope
5. Immersion oil
6. 24 hour grown culture of organism
7. Crystal violet
8. Gram’s iodine
9. Ethyl alcohol
10. Safranin

Procedure

1. Take a clean glass slide, add a loopful of culture sample.
2. Allow smear to air dry, then heat fix the slide.
3. Flood the slide with primary stain i.e., crystal violet for 1 min.³
4. Rinse the slide under the running tap water.
5. Flood the slide with mordant i.e., gram’s iodine for 1 min.²
6. Rinse under the running tap water.
7. Flood the slide with ethyl alcohol i.e., decolourizer for 10 to 15 sec. ²
8. Rinse under the running tap water.
9. Flood the slide with secondary stain i.e., safranin for 30 sec.³
10. Rinse under the running tap water.
11. Allow it to air dry, then observe under the oil immersion lens of microscope.

Conclusion

In the culture sample purple stain organism appear as gram positive and pink stain organism appear as gram negative.

Acid fast staining

Acid fast staining also known as Ziel-Nehlson method.

Requirement

1. Clean Glass slide
2. Inoculation loop
3. Bunsen burner
4. Microscope
5. Immersion oil
6. 24 hour grown culture of organism
7. Carbol fuschin
8. Methylene blue
9. Ethyl alcohol
10. Water bath

Procedure

1. Take a clean glass slide, add loopful of culture sample.
2. Allow it to air dry, and heat fix the smear.
3. Flood the slide with carbol fuschin and heat it carefully on water bath until steam rises, keep it for 3-5 min. ²
4. Rinse under the running tap water.
5. Flood slide with alcohol for 10-15 sec.¹
6. Rinse under the running tap water.
7. Flood slide with the methylene blue for 1 min.²
8. Rinse under the running tap water.
9. Allow to air dry, observe under the oil immersion lens (100x).

Conclusion

The acid-fast bacteria appear in red colour and non-acid fast bacteria appear in blue in the provided culture sample.

Endospore Staining

Bacterial species create endospores, which are exceptionally heat resistant structures. The most common used method is malachite green-Schaeffer and Fulton.

Requirement

1. Clean Glass slide
2. Inoculation loop
3. Bunsen burner
4. Microscope
5. Immersion oil
6. 24 hour grown culture of organism
7. Malachite green
8. Safranin
9. Paper towel
10. Water bath

Procedure

1. Take a clean glass slide, add loopful of culture sample.
2. Allow smear to air dry.
3. Place slide on water bath using staining rack.⁴
4. Cover the slide with piece of paper towel, flood slide with malachite green for 5 min, and keep saturated with stain at the time of heating.¹
5. Rinse under running tap water.
6. Flood slide with counter stain i.e., safranin for 30 sec. ¹
7. Rinse under tap water.
8. Allow to air dry, observe under oil immersion lens(100x).

Conclusion

The vegetative cells appear in pink and spores appear in green colour.

Capsule Staining

The presence of the capsule can be easily seen using a differential stain called a capsule stain, which uses acidic and basic dyes to stain the bacterial cells and background, respectively. The bacteria are encased in the capsule, which is produced in the cytoplasm and secreted outside the cell.

Requirement

1. Clean Glass slide
2. Inoculation loop
3. Bunsen burner
4. Microscope
5. Immersion oil
6. 24 hour grown culture of organism
7. India ink
8. Crystal violet

Procedure

1. Take a clean glass slide, add a drop of India ink at the end of the slide.¹
2. Take loopful of culture and mix with the stain.
3. Spread the drop out over the slide by using the edge of another slide.¹
4. Allow it to air dry, and heat fix the slide.
5. Flood the slide with the crystal violet for 1 min.⁴
6. Rinse under the tap water.
7. Air dry slide, observe under oil immersion lens.

Conclusion

The purple cells surrounded by a clear halo dark background. The halo is the capsule.

Flagella Staining

Flagella staining is used to determine if a bacterium is motile or not. Based on the structure, arrangement, size, and location of the bacterial flagella, this particular staining method aids in the distinction of the genus and species.

Requirements

1. Clean Glass slide
2. Inoculation loop
3. Bunsen burner
4. Microscope
5. Immersion oil
6. 24 hour grown culture of organism
7. Cover slip
8. Distilled water
9. Ryu stain

Procedure

1. Take loopful of culture, add it to the distilled water keep it for 10-20 min.
2. Add a drop of water containing culture.
3. Place a cover slip over the drop of the sample.¹
4. Observe the slide under the 40x lens, if the motile cells are seen.
5. Add 1-2 drops of the Ryu stain at edge of the cover slip ⁴
6. Keep slide at room temperature with stain for 10 min.
7. Observe the slide under oil immersion lens(100x).

Conclusion

The purple colour flagella cells are observed.

Lactophenol Cotton Blue (LPCB) Mounts

A mounting media and staining agent used in the creation of slides for microscopic investigation of fungus is lactophenol cotton blue solution.

Requirement

1. Clean Glass slide
2. Inoculation loop
3. Bunsen burner
4. Microscope
5. Lactophenol cotton blue
6. Culture of organism
7. Cover slip
8. Dissecting needles

Procedure

1. Take a clean glass slide, add drop of lactophenol cotton blue stain.⁵
2. Add fungal specimen over the drop of stain.
3. With the help of sterile dissecting needles, gently tease the fungal sample.
4. Place cover slip over the sample, avoid air bubble and gaps.⁴
5. Observe under the microscope.

Conclusion

The blue colour-stained fungal structure against the pale blue background.

[ps2id id=’references’ target=”/][ps2id id=’1′ target=”/]

REFERENCES

1. Microbes in practice, Bisen Prakash S., IK International, New Delhi, 2014, 139-155.
2. staining_techniques_in_microbiology..pdf
3. https://vlab.amrita.edu/index.php?sub=3&brch=73&sim=208&cnt=1
4. Types of Staining Techniques Used in Microbiology – Microbe Online
5. Lactophenol Cotton Blue Staining Principle, Procedure, Result. (microbiologynote.com)

Also read:

• Affinity Purification of Immunoglobulin
• Preparation of enzyme conjugated antibodies
• Purification of Immunoglobulin by Precipitation
• Microplate based Alamar Blue Assays
• Isolation of Genomic DNA from E. coli
• Estimation of DNA and its purity check using UV spectrophotometer (A260 measurement)
• Study of Polyploidy in Onion Root Tip Bycolchicine Treatment

FAQs

Q1    Why should the slide be flooded with stain at the time of heating?

Ans.  For uniform distribution of heat, otherwise slide may break.

Q2   Is there any alternative for heat fixation of smear?

Ans. Yes, there is use of chemicals for smear fixation like aldehydes, osmium tetroxide, Acetic acid.

 

🔴 Would you like to attempt Labmonk Daily quiz? Click here

🔵 Check out Jobs & Exam Notices. Labmonk Notice Board

🔴 Labmonk Scholarships. Click here

🔵 Labmonk Blog. Click here

🔴 Do you need notes? Click here

Watch Career related videos on Youtube: Watch now !!