Methylene Blue Stain: Introduction, Principle, Composition, Preparation, Staining Requirements, Staining Procedure, Result Interpretation, and Keynotes

Introduction

Methylene Blue Stain, also known as methylene blue aqueous is an ordinary stain. It is used for capsule stain as well as simple staining for a rapid and easy way to determine cell shape, size, and arrangements of bacteria. It is also made easier to differentiate bacteria from yeast cells and presumptively identify the bacterial isolate by a simple wet mount method.

Methylene blue stain and microbial growth plates
Fig. Methylene blue stain and microbial growth plates

Principle of Methylene Blue Stain

The capsule is distinct from the slime layer that most bacterial cells form in that it is a thick, detectable, discrete layer outside the cell wall. The capsule stain takes on an acidic stain while a basic stain detects capsule production. A positive capsule stain requires a mordant that precipitates the capsule. By counterstaining with dyes like methylene blue or safranin or crystal violet, the bacterial cell wall takes up the dye. Capsules of organisms appear colorless with stained cells against a dark background.

Cryrptococcus neoformans capsules in Methylene blue wet mount Microscopy
Fig. Cryptococcus neoformans capsules in Methylene blue wet mount Microscopy

Composition of Methylene Blue Stain

  • Methylene blue 0.5 gm
  • Distilled water 100.0 ml

Preparation of Methylene Blue Stain

  • Weigh 0.5 gm methylene blue powder on a piece of clean paper (pre-weighed) and dissolve the stain in about 30 ml of water.
  • Finally, add the remaining water.
  • Transfer the stain to a clean brown bottle.
  • Label the bottle and store it in a dark place at room temperature.
  • The stain is usable for several months.

Requirements for Staining Procedure 

  • Methylene blue (aqueous)
  • Clean and grease-free glass slides
  • Test organisms/ Sample (Clinical samples; food and dairy samples, Water samples)
  • Bunsen burner/ Sprit lamp
  • Inoculating loop or sterile bamboo stick
  • Compound Microscope
  • Cedarwood oil
  • Waste bin
  • Gloves
  • Control strain (Encapsulated bacteria/ fungus)

Staining Procedure of Methylene Blue Stain

For Capsule staining

  1. Transfer aseptically a loopful of microbial culture on a clean and grease-free dry slide.
  2. Mix it with a loopful of aqueous methylene blue.
  3. Prepare a smear by using a glass slide.
  4. Leave the smear to air dry slowly.
  5. Examine the entire smear systematically with the low power objective (10X ) and low light intensity.
  6.  If any suspicious capsule is encountered, examine it with the oil immersion objective (100X).

Result Interpretation of Methylene Blue Stain

  • The capsule of Microbes: Colourless (hollow bodies)
  • Background: Bluish
  • Control strain: clear halos surrounding the cells

Limitations of  Methylene Blue Stain

  1. Capsules are more delicate since using heat during the staining process should be avoided given that capsules would be easily destroyed.
  2. Washing the slide with water is also avoided in capsule staining since it would dislodge the capsule.

Keynotes on Methylene Blue Stain

  1. Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by heating.
  2. A drop of serum can be applied during smearing to enhance the size of the capsule which makes it easier observation with a typical compound light microscope.
  3. A capsule is a gelatinous outer layer secreted by the cell of the organism ( bacteria/ fungi) that surrounds and adheres to the cell wall. Most capsules are composed of polysaccharides, but some are composed of polypeptides.
  4. The capsule stain begins as a negative stain. Cells are spread in a film of the acidic stain and are not heat-fixed. Heat-fixing causes shrinkage of the cells, leaving an artefactual white halo around them that might be falsely interpreted as a capsule.
  5. Simple staining has only one stain or dye. Methylene blue in simple staining gives up a hydroxide ion which leaves the stain positively charged. Since the surface of most bacterial cells is negatively charged, these positively charged stains adhere readily to the cell surface.
  6. Methylene blue is a good nuclear stain, the staining solutions are always either aged or alkalized, if not both. More importantly, it is an excellent stain for ribonucleic acid (RNA), which is the important stainable constituent of bacterial bodies, lymphocytes, hemoprotozoal cytoplasm, and of nerve cell tigroid granules.
  7. Loeffler’s alkaline methylene blue stain and Loeffler’s alkaline methylene blue are the modification of the original methylene blue stains. They are applied in In Ziehl-Neelsen Staining, and for the expression of metachromatic granules of C. diphtheriae.
  8. New methylene blue is the preferred stain for the reticulofilamentous material and it is chemically different from methylene blue since methylene blue is a poor reticulocyte stain.

Methylene blue stained Footage

Klebsiella pneumoniae capsules in methylene blue stained smear

Klebsiella pneumoniae capsules in methylene blue stained smear
Fig. Klebsiella pneumoniae capsules in methylene blue-stained smear

Bacillus species in Methylene blue wet mount microscopy

Bacillus species in Methylene blue wet mount microscopy
Fig. Bacillus species in Methylene blue wet mount microscopy

Live and dead yeast cells of Cryptococcus neoformans in Loeffler’s methylene blue

Live and dead yeast cells of Cryptococcus neoformans in Loeffler's methylene blue
Fig. Live and dead yeast cells of Cryptococcus neoformans in Loeffler’s methylene blue

Giardia cysts in methylene blue wet mount of stool/ feces microscopy

Giardia cysts in methylene blue wet mount of stool/ feces microscopy
Fig. Giardia cysts in methylene blue wet mount of stool/ feces microscopy

White blood cell in new methylene blue wet mount of blood microscopy

White blood cell in new methylene blue wet mount of blood microscopy
Fig. White blood cell in new methylene blue wet mount of blood microscopy

Acid-fast bacilli of Mycobacterium tuberculosis in Ziehl-Neelsen stained smear of sputum with Loeffler’s methylene blue counterstain

Acid fast bacilli of Mycobacterium tuberculosis in Ziehl-Neelsen stained smear of sputum with Loeffler's methylene blue counterstain
Fig. Acid-fast bacilli of Mycobacterium tuberculosis in Ziehl-Neelsen stained smear of sputum with Loeffler’s methylene blue counterstain

Further Readings

  1. Methylene blue -Sigma-Aldrich
  2. Jorgensen,J.H., Pfaller , M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
  3. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
  4. Downes F. P. and Ito K. (Ed.), 2001, Compendium of Methods for the Microbiological Examination of Foods, 4th ed., APHA, Washington, D.C

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  76. Great article. It is unfortunate that over the last decade, the travel industry has already been able to to fight terrorism, SARS, tsunamis, bird flu virus, swine flu, as well as the first ever real global tough economy. Through everything the industry has really proven to be effective, resilient plus dynamic, discovering new solutions to deal with adversity. There are always fresh difficulties and opportunities to which the marketplace must just as before adapt and reply.

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  80. Thanks for your posting on the travel industry. I would also like to add that if you are a senior taking into consideration traveling, it’s absolutely crucial that you buy travel insurance for retirees. When traveling, seniors are at biggest risk of getting a health emergency. Receiving the right insurance cover package for the age group can safeguard your health and provide peace of mind.

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  81. Thanks for your short article. I would also love to say that the health insurance agent also works for the benefit of the particular coordinators of a group insurance policy. The health insurance professional is given an index of benefits looked for by anyone or a group coordinator. What any broker does indeed is look for individuals as well as coordinators that best match up those demands. Then he reveals his referrals and if both parties agree, this broker formulates a contract between the 2 parties.

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  101. Thanks for enabling me to gain new concepts about personal computers. I also hold the belief that certain of the best ways to help keep your mobile computer in prime condition is to use a hard plastic-type case, or perhaps shell, that fits over the top of one’s computer. A lot of these protective gear are generally model specific since they are made to fit perfectly within the natural covering. You can buy all of them directly from the vendor, or through third party sources if they are for your laptop, however don’t assume all laptop could have a shell on the market. Just as before, thanks for your recommendations.

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  103. Thanks for sharing your ideas on this blog. Likewise, a misconception regarding the banking institutions intentions while talking about foreclosure is that the traditional bank will not have my payments. There is a certain quantity of time that this bank is going to take payments occasionally. If you are also deep inside hole, they may commonly call that you pay that payment fully. However, that doesn’t mean that they will not take any sort of payments at all. Should you and the financial institution can have the ability to work something out, this foreclosure practice may stop. However, when you continue to miss out on payments wih the new strategy, the foreclosure process can pick up exactly where it left off.

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  111. It is indeed my belief that mesothelioma is most dangerous cancer. It has unusual properties. The more I really look at it the more I am persuaded it does not react like a real solid cells cancer. In case mesothelioma can be a rogue virus-like infection, so there is the chance for developing a vaccine along with offering vaccination to asbestos subjected people who are really at high risk associated with developing foreseeable future asbestos linked malignancies. Thanks for discussing your ideas about this important health issue.

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  114. Thanks for giving your ideas on this blog. In addition, a myth regarding the banking companies intentions any time talking about foreclosure is that the financial institution will not have my installments. There is a fair bit of time which the bank requires payments in some places. If you are too deep in the hole, they will commonly call that you pay the particular payment in full. However, that doesn’t mean that they will have any sort of repayments at all. In case you and the lender can seem to work anything out, the particular foreclosure procedure may halt. However, if you ever continue to skip payments under the new approach, the property foreclosures process can pick up where it was left off.

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  115. Thanks for your text. I would also love to say that a health insurance broker also works best for the benefit of the coordinators of the group insurance. The health broker is given a list of benefits desired by somebody or a group coordinator. Exactly what a broker can is try to find individuals or coordinators which will best match up those requirements. Then he gifts his referrals and if the two of you agree, this broker formulates a contract between the two parties.

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  116. In accordance with my research, after a in foreclosure process home is sold at a sale, it is common for the borrower in order to still have any remaining unpaid debt on the mortgage loan. There are many creditors who seek to have all expenses and liens cleared by the subsequent buyer. Nevertheless, depending on certain programs, restrictions, and state laws and regulations there may be many loans that are not easily solved through the shift of personal loans. Therefore, the responsibility still rests on the consumer that has obtained his or her property foreclosed on. Many thanks for sharing your ideas on this blog.

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  118. Thanks for your posting on the traveling industry. I might also like to add that if you’re a senior thinking about traveling, it’s absolutely vital that you buy traveling insurance for older persons. When traveling, retirees are at greatest risk of experiencing a professional medical emergency. Obtaining the right insurance cover package in your age group can look after your health and provide you with peace of mind.

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  123. Thanks for revealing your ideas. I’d personally also like to express that video games have been at any time evolving. Modern technology and innovative developments have assisted create authentic and fun games. These kinds of entertainment video games were not actually sensible when the concept was first of all being tried. Just like other styles of technological know-how, video games also have had to develop as a result of many years. This itself is testimony on the fast development of video games.

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  124. One more thing. It’s my opinion that there are quite a few travel insurance web sites of trustworthy companies that allow you enter your vacation details and have you the insurance quotes. You can also purchase the international travel cover policy online by using the credit card. All you should do is always to enter your current travel details and you can see the plans side-by-side. Just find the system that suits your allowance and needs and then use your bank credit card to buy it. Travel insurance on the web is a good way to begin looking for a dependable company with regard to international holiday insurance. Thanks for discussing your ideas.

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  125. Thanks for your valuable post. In recent times, I have been able to understand that the particular symptoms of mesothelioma cancer are caused by a build up of fluid relating to the lining on the lung and the breasts cavity. The disease may start within the chest area and distribute to other parts of the body. Other symptoms of pleural mesothelioma cancer include weight reduction, severe respiration trouble, temperature, difficulty eating, and infection of the neck and face areas. It should be noted that some people existing with the disease never experience virtually any serious indications at all.

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  126. I have seen many useful points on your web-site about pcs. However, I’ve the thoughts and opinions that notebook computers are still not quite powerful sufficiently to be a wise decision if you normally do things that require loads of power, such as video modifying. But for website surfing, word processing, and the majority of other frequent computer work they are just great, provided you don’t mind the screen size. Thanks for sharing your thinking.

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  130. Thanks for the helpful article. It is also my opinion that mesothelioma has an particularly long latency period of time, which means that signs and symptoms of the disease would possibly not emerge right until 30 to 50 years after the 1st exposure to asbestos. Pleural mesothelioma, which can be the most common form and is affecting the area throughout the lungs, will cause shortness of breath, breasts pains, as well as a persistent coughing, which may bring about coughing up blood vessels.

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  131. Thanks for your information on this blog. Just one thing I want to say is that often purchasing gadgets items from the Internet is not new. In fact, in the past decades alone, the marketplace for online electronic products has grown drastically. Today, you could find practically just about any electronic system and devices on the Internet, from cameras plus camcorders to computer pieces and gaming consoles.

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  137. One important issue is that when you are searching for a student loan you may find that you’ll want a cosigner. There are many conditions where this is true because you could find that you do not employ a past credit standing so the financial institution will require that you’ve got someone cosign the borrowed funds for you. Great post.

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  138. In accordance with my observation, after a the foreclosure home is bought at a sale, it is common for the borrower in order to still have a remaining balance on the mortgage. There are many loan providers who aim to have all expenses and liens paid off by the up coming buyer. Even so, depending on certain programs, regulations, and state laws there may be several loans which aren’t easily solved through the switch of personal loans. Therefore, the duty still lies on the debtor that has got his or her property in foreclosure process. Thank you sharing your thinking on this blog site.

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  139. A further issue is really that video gaming has become one of the all-time most significant forms of excitement for people spanning various ages. Kids enjoy video games, and adults do, too. The particular XBox 360 is probably the favorite games systems for individuals that love to have hundreds of video games available to them, as well as who like to play live with others all over the world. Thank you for sharing your thinking.

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  142. I’d personally also like to state that most people who find themselves with no health insurance can be students, self-employed and those that are laid-off. More than half from the uninsured are under the age of Thirty-five. They do not think they are requiring health insurance as they are young as well as healthy. Its income is often spent on housing, food, as well as entertainment. Some people that do work either full or in their free time are not provided insurance by their jobs so they proceed without owing to the rising valuation on health insurance in america. Thanks for the ideas you discuss through this site.

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  169. 2024娛樂城的創新趨勢

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  170. Деревянные дома под ключ
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  171. Дома АВС – Ваш уютный уголок

    Мы строим не просто дома, мы создаем пространство, где каждый уголок будет наполнен комфортом и радостью жизни. Наш приоритет – не просто предоставить место для проживания, а создать настоящий дом, где вы будете чувствовать себя счастливыми и уютно.

    В нашем информационном разделе “ПРОЕКТЫ” вы всегда найдете вдохновение и новые идеи для строительства вашего будущего дома. Мы постоянно работаем над тем, чтобы предложить вам самые инновационные и стильные проекты.

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  173. Understanding the processes and protocols within a Professional Tenure Committee (PTC) is crucial for faculty members. This Frequently Asked Questions (FAQ) guide aims to address common queries related to PTC procedures, voting, and membership.

    1. Why should members of the PTC fill out vote justification forms explaining their votes?
    Vote justification forms provide transparency in decision-making. Members articulate their reasoning, fostering a culture of openness and ensuring that decisions are well-founded and understood by the academic community.

    2. How can absentee ballots be cast?
    To accommodate absentee voting, PTCs may implement secure electronic methods or designated proxy voters. This ensures that faculty members who cannot physically attend meetings can still contribute to decision-making processes.

    3. How will additional members of PTCs be elected in departments with fewer than four tenured faculty members?
    In smaller departments, creative solutions like rotating roles or involving faculty from related disciplines can be explored. Flexibility in election procedures ensures representation even in departments with fewer tenured faculty members.

    4. Can a faculty member on OCSA or FML serve on a PTC?
    Faculty members involved in other committees like the Organization of Committee on Student Affairs (OCSA) or Family and Medical Leave (FML) can serve on a PTC, but potential conflicts of interest should be carefully considered and managed.

    5. Can an abstention vote be cast at a PTC meeting?
    Yes, PTC members have the option to abstain from voting if they feel unable to take a stance on a particular matter. This allows for ethical decision-making and prevents uninformed voting.

    6. What constitutes a positive or negative vote in PTCs?
    A positive vote typically indicates approval or agreement, while a negative vote signifies disapproval or disagreement. Clear definitions and guidelines within each PTC help members interpret and cast their votes accurately.

    7. What constitutes a quorum in a PTC?
    A quorum, the minimum number of members required for a valid meeting, is essential for decision-making. Specific rules about quorum size are usually outlined in the PTC’s governing documents.

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  174. One other thing I would like to state is that instead of trying to match all your online degree tutorials on days of the week that you finish off work (as most people are tired when they go back home), try to have most of your lessons on the weekends and only a few courses in weekdays, even if it means a little time away from your saturdays. This is really good because on the saturdays and sundays, you will be extra rested as well as concentrated for school work. Thx for the different points I have mastered from your blog.

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