Lactophenol Cotton Blue (LPCB) Stain: Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, Uses of LPCB Stain, Keynotes, and LPCB Stained Footages

Introduction of Lactophenol Cotton Blue (LPCB) Stain

LPCB stands for lactophenol cotton blue and Lactophenol Cotton Blue (LPCB) Stain is a combination of fixative, staining, and clearing agent. LPCB stain uses both as a mounting fluid and a stain and it is used for staining and microscopic identification of fungi. Its contents are lactic acid, phenol, cotton blue, and glycerol. Lactic acid helps in preserving the morphology of the fungal elements. Phenol acts as a disinfectant. Cotton blue stains the fungal elements as well as intestinal parasitic (cyst, ova, and oocyst) and non-parasitic structures (vegetable cells, mucus, muscle fibers, and other artifacts) while glycerol is a hygroscopic agent that prevents drying.

Lactophenol Cotton Blue (LPCB) Stain: Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, Uses of LPCB Stain, Keynotes and LPCB Stained Footages
Fig. Lactophenol Cotton Blue (LPCB) Stain, fungal growth, and LPCB prepared slide for Microscopy

Principle of Lactophenol Cotton Blue (LPCB) Stain

Ingredients of lactophenol cotton blue (LPCB) stain-like lactic acid act as a clearing agent and aid in preserving the fungal structures. Similarly, phenol kills the organism and fixes it while glycerol prevents drying. Cotton blue stains the chitin in the cell wall of fungi and identification of filamentous fungi is made by their characteristic microscopic morphology e.g. shape, size, arrangement of spores, and hyphae providing color to the structure. It can be used alone or in conjunction with KOH.

Composition of Lactophenol Cotton Blue (LPCB) Stain

For 200 ml
Lactic acid: 40 ml
Phenol: 40 ml
Glycerol:80 ml
Cotton blue (Poirier blue or Aniline blue): 0.1 g
Distilled water: 40 ml

LPCB Stain Preparation

  • Dissolve phenol in lactic acid, glycerol, and distilled water.
  • Ultimately, add cotton blue and mix well.
  • But this LPCB stain is prepared over two days.
  • On the first day, dissolve the cotton blue in distilled water and leave it overnight to get rid of the insoluble dye.
  • On the second day, wearing gloves add the phenol crystals to the lactic acid in a glass beaker. Put on a magnetic stirrer until the phenol is dissolved or do manually.
  • Add the glycerol.
  • Filter the cotton blue and distilled water (D/W) solution into the phenol/glycerol/ lactic acid solution.
  • Mix and store at room temperature.

Requirements for Lactophenol Cotton Blue (LPCB) Staining

  • Compound light microscope
  • LPCB stain
  • Clean and grease-free microscopic slides
  • Coverslip
  • Dropper or bamboo sticks
  • Fungal growth  in the medium

The procedure of LPCB Preparation

  • Take a clean and grease-free glass slide.
  • Put a large drop of LPCB with a Pasteur pipette or dropper.
  •  Transfer a small quantity of the culture to the drop.
  • Tease the culture (in case of a mold) well with teasing needles
    so as to get a uniform spread.
  • Put on a coverslip gently to avoid entrapment by air bubbles.
  •  Examine under low- (10 X) and high-power (40 X) objectives.
  • Observe the morphological features carefully as shown below.

Observation

Fungal structures appear dark blue.

Results and interpretations of  Lactophenol Cotton Blue (LPCB) Stain

Different fungi under LPCB wet mount will show different types of morphological structures including hyphae and spores

  • Fungal spores, hyphae, and fruiting structures: Takes stained blue
  • Background: stains pale blue.

Application of Lactophenol Cotton Blue (LPCB) Stain

  •  For staining and microscopic identification of fungi observing fungal spores, hyphae, and fruiting structures.
  • It is also applicable in parasitology for the observation of Cysts of intestinal protozoa and ova take blue color while ova of helminths are stained deep blue.

Keynotes on LPCB Stain

  1. LPCB wet mount is always examined at least 30 minutes after preparation.
  2. A wet mount preparation should neither be too thick nor too thin.
  3.  In this preparation, both bile-stained and non-bile-stained helminthic eggs are stained blue.
  4. LPCB kills the trophozoites of Entamoeba and Trichomonas, hence, can not be demonstrated by this.
  5. In LPCB wet mount of stool phenol and lactic clear fecal debris.
  6. In the LPCB wet mount of stool, glycerol provides a semi-permanent preparation. Cyst of intestinal protozoa and ova takes blue color while ova of helminths are stained deep blue. An additional advantage of this stain is that it can also detect blue-colored Cyclospora and Isospora oocyst.

Limitations of LPCB Stain

Even though LPCB stain is very useful has some demerits-

  1. LPCB stain is only applicable for the presumptive identification method of fungi.
  2. The ingredient of the LPCB solution may disrupt the original morphology of the fungi.
  3. The stain can only be used to identify mature fungi and their structures and not the young vegetative forms of fungi.
  4. A wet mount preparation should neither be too thick nor too thin.
  5. Application of LPCB stain in Parasitology is not preferred because it kills the trophozoites of Entamoeba and Trichomonas.
  6. This LPCB stain has an expiry date and thus can only use before expiry.

LPCB Stained Footages

Cryptococcus neoformans in LPCB Tease mount Microscopy

Cryptococcus neoformans in LPCB Tease mount Microscopy
Fig. Cryptococcus neoformans in LPCB Tease mount Microscopy

Geotrichum candidum in LPCB tease mount showing arthroconidia

Geotrichum candidum in LPCB tease mount showing arthroconidia
Fig. Geotrichum candidum in LPCB tease mount showing arthroconidia

Malassezia in LPCB tease mount preparatiion Microscopy

Malassezia in LPCB tease mount preparatiion Microscopy
Fig. Malassezia in LPCB tease mount preparatiion Microscopy

Aspergillus structures like septate hyphae, conidiophore or stipe, vesicle, Phialides, conidia, metulae, dichotomous branching (at an angle of approximately 45°) in LPCB Tease Mount Microscopy

Aspergillus structures like septate hyphae, conidiophore or stipe, vesicle, Phialides, conidia, metulae, dichotomous branching (at an angle of approximately 45°) in LPCB Tease Mount Microscopy
Fig. Aspergillus structures like septate hyphae, conidiophore or stipe, vesicle, Phialides, conidia, metulae, dichotomous branching (at an angle of approximately 45°) in LPCB Tease Mount Microscopy

Aspergillus fumigatus mycelium and conidia in LPCB-stained Microscopic Footage

Aspergillus fumigatus mycellium and conidia in LPCB stained Microscopic Footage
Fig. Aspergillus fumigatus mycelium and conidia in LPCB-stained Microscopic Footage

Further Readings on  LPCB Stain

  1. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
  2.  Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
  3. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmons, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Francisco 1996.
  4. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  5. A Text-Book of Medical Mycology. Editor: Jagdish Chander.  Publication Mehata, India.
  6. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1706009/
  8. https://mycology.adelaide.edu.au/laboratory/lacto/
  9. http://himedialabs.com/TD/S016.pdf

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  74. 2024娛樂城的創新趨勢

    隨著2024年的到來,娛樂城業界正經歷著一場革命性的變遷。這一年,娛樂城不僅僅是賭博和娛樂的代名詞,更成為了科技創新和用戶體驗的集大成者。

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  80. Overview of Digital Currency Deal Validation and Conformity Solutions

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    항공과 해운 수출 건에 대한 허가서를 효율적으로 발급받는 방법을 상세히 안내하며, 고객의 요청에 따라 이메일로 전달됩니다.

    노데이터 처리 안내
    운송장 번호 없는 주문에 대한 새로운 처리 방안을 도입하여, 노데이터 발생 시 관리비가 부과되지만 서비스 품질을 개선합니다.

    5. 고객과의 소통
    카카오톡 상담: 실시간 상담을 통해 고객의 궁금증을 해결합니다.
    공지사항 알림: 서비스 이용 중 필수 정보를 지속적으로 업데이트합니다.
    메인 서비스는 고객 만족을 최우선으로 하며, 지속적인 개선과 세심한 관리를 통해 최상의 경험을 제공합니다.

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  82. 일본 소비세 환급, 네오리아와 함께라면 간편하고 안전하게

    일본 소비세 환급은 복잡하고 까다로운 절차로 많은 구매대행 셀러들이 어려움을 겪는 분야입니다. 네오리아는 다년간의 경험과 전문성을 바탕으로 신뢰할 수 있는 서비스를 제공하며, 일본 소비세 환급 과정을 쉽고 효율적으로 처리합니다.

    1. 일본 소비세 환급의 필요성과 네오리아의 역할
    네오리아는 일본 현지 법인을 설립하지 않아도 합법적인 방식으로 소비세 환급을 받을 수 있는 솔루션을 제공합니다. 이를 통해:

    한국 개인사업자와 법인 사업자 모두 간편하게 환급 절차를 진행할 수 있습니다.
    일본의 복잡한 서류 심사를 최소화하고, 현지 로컬 세리사와 협력하여 최적의 결과를 보장합니다.
    2. 소비세 환급의 주요 특징
    일본 연고가 없어도 가능: 일본에 사업자가 없더라도 네오리아는 신뢰할 수 있는 서비스를 통해 소비세 환급을 지원합니다.
    서류 작성 걱정 해결: 잘못된 서류 제출로 환급이 거절될까 걱정될 필요 없습니다. 네오리아의 전문 대응팀이 모든 과정을 정밀하게 관리합니다.
    현지 법인 운영자를 위한 추가 지원: 일본 내 개인사업자나 법인 운영자에게는 세무 감사와 이슈 대응까지 포함된 고급 서비스를 제공합니다.
    3. 네오리아 서비스의 장점
    전문성과 신뢰성: 정부로부터 인정받은 투명성과 세무 분야의 우수한 성과를 자랑합니다.
    맞춤형 서포트: 다양한 사례를 통해 쌓은 경험으로 고객이 예상치 못한 어려움까지 미리 해결합니다.
    로컬 업체에서 불가능한 고급 서비스: 한국인 고객을 위해 정확하고 간편한 세무회계 및 소비세 환급 서비스를 제공합니다.
    4. 네오리아가 제공하는 혜택
    시간 절약: 복잡한 절차와 서류 준비 과정을 전문가가 대신 처리합니다.
    안심 환급: 철저한 관리와 세심한 대응으로 안전하게 환급을 받을 수 있습니다.
    추가 서비스: 세무감사와 이슈 발생 시 즉각적인 지원으로 사업의 연속성을 보장합니다.
    네오리아는 소비세 환급이 복잡하고 어렵다고 느껴지는 고객들에게 최적의 길잡이가 되어드립니다. 신뢰를 바탕으로 한 전문적인 서비스로, 더 이상 소비세 환급 문제로 고민하지 마세요!

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  83. I was wondering if you ever considered changing the layout
    of your site? Its very well written; I love what youve got
    to say. But maybe you could a little more in the way of content so people could
    connect with it better. Youve got an awful lot of text for only having 1 or
    two images. Maybe you could space it out better?

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  84. I do not know if it’s just me or if everybody else experiencing problems with your blog.

    It looks like some of the written text in your posts are running
    off the screen. Can somebody else please comment and
    let me know if this is happening to them too? This may be a problem with my web browser because
    I’ve had this happen previously. Thank you

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  85. I love your blog.. very nice colors & theme. Did you make this website yourself or did you hire someone to do it for you? Plz answer back as I’m looking to create my own blog and would like to find out where u got this from. thanks a lot

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