Stain in Microbiology for Specimen Processing: Name List, Introduction

Stain in Microbiology for Specimen Processing: Name List, Introduction and Uses

Stain in Microbiology

Stain in Microbiology chapter contains nearly all the stains those applicable in microbiology that contains name list of stains, introduction, uses, in some principle, procedure, result in interpretation, keynotes, etc.

Stain list

Acridine Orange Stain
Auramine-Rhodamine Stain
Calcofluor White Stain
Cryptosporidium Stain
Direct Fluorescent-Antibody Stain
Dobell & O’Connor Iodine
Flagella Stain
Giemsa Stain
Gram Stain
India Ink Stain (Nigrosin)
Iron Hematoxylin Stain
Kinyoun Stain for AFB
Lactophenol Cotton Blue
Lugol’s Iodine Stain
Methenamine Silver Stain
Methylene Blue Stain
Periodic Acid-Schiff (PAS) Stain
Sudan Black B Stain
Sudan Black IV Stain
Trichrome Stain
Uristain
Wright Stain
Ziehl-Neelsen Stain for AFB

1. Acridine Orange Stain

Introduction of Acridine orange

Acridine orange is a fluorochrome stain and uses the rapid identification of Trichomonas vaginalis, yeast cells, and clue cells in vaginal smears. It can also use to detect intracellular gonococci, meningococci, and other bacteria, particularly in blood cultures.

Principle of Acridine orange

Acridine orange is a fluorochrome that causes DNA to fluoresce green and
RNA to fluoresce orange-red.

The requirement for Acridine orange

Acridine orange acid stain
Alcohol saline solution
physiological saline
Test specimen
Glass Slide
Inoculating loop or sterile bamboo stick
Fluorescence microscope having a BG 12 exciter filter and No. 44 and No. 53 barrier filters

The procedure of Acridine orange staining

Cover the unfixed dried smear with the acridine orange acid stain for 5–10 seconds and smear fixes due to being fixative is contained in the stain.
Now, wash off the stain, and decolorize the smear with alcohol saline solution for 5–10 seconds.
Rinse the smear with physiological saline, and place the slide in a draining rack.
Add a drop of saline or distilled water to the smear, and cover with a cover glass.

Observation

Examine first with the 10X objective to see the distribution of fluorescing material, and then with the 40X objective to identify Trichomonas vaginalis and to detect yeast cells, bacteria, and parasites( chromatoid bars of Entamoeba histolytica/ dispar).

Results interpretation of Acridine orange

Trichomonas vaginalis : Orange-red with the yellow-green nucleus

Yeast cells: Orange

Bacteria: Orange

Pus cells ( WBCs): Yellow-green
Epithelial cells: Yellow-green

Keynotes

The appearance of chromatoid bars of Entamoeba histolytica/ dispar in an acridine-stained smear is shown in this video clip as well as the above image.
In bacterial vaginosis, the orange staining bacteria adhering to the green epithelial cells (clue cells) can be clearly seen.

Acridine orange acid stain preparation

To make 100 ml
Acridine orange: 26 mg
Acetic acid, glacial (concentrated): 2 ml

Distilled water: 98 ml

Distilled water (D/W), acridine orange, and transfer to a
brown bottle of 100 ml capacity.
Fill a cylinder to the 98 ml mark with distilled water.
Add 2 ml of glacial acetic acid, i.e. to the 100 ml mark.
Add about half of the acid solution to the acridine orange, and mix until the dye is completely dissolved.
Add the remaining acid solution, and mix well.
Label the bottle, and store it at room temperature.
The stain is stable for several weeks.
Caution: Glacial acetic acid is a corrosive chemical with an irritating vapor, therefore handle it with care in a well-ventilated room.

Alcohol saline solution

To make 250 ml

Ethanol or methanol, absolute: 5 ml
Physiological saline or Sodium chloride,

0.85 g/dl i.e 0.85% w/v : 245 ml

Fill a cylinder (250 ml capacity) to the 245 ml mark with the saline solution.
Add 5 ml of absolute ethanol or methanol to the 250 ml mark.
Transfer to a screw-cap bottle, and mix well.
Label the bottle, and store it in a cool place.
The reagent is stable for several months.

Caution: Ethanol and methanol are highly flammable, therefore use these chemicals well away from an open flame.

Physiological saline, 8.5 g/l (0.85% w/v)

To make 1 liter
Sodium chloride: 8.5 g
Distilled water: up to 1 liter

Weigh the sodium chloride, and transfer it to a leak-proof bottle premarked to hold 1 liter.
Add distilled water to the 1-liter mark, and mix until the salt is fully dissolved.
Label the bottle, and store it at room temperature.
The reagent is stable for several months. Discard if it becomes contaminated.

2. Auramine-Rhodamine Stain

Introduction of auramine -rhodamine stain

It is a fluorochrome stain and used to visualize acid-fast structures of various microorganisms especially Mycobacterium tuberculosis and in modified form for Mycobacterium leprae , Nocardia species, Cryptosporidium parvum, Cyclospora cayetanensis , Isospora belli and fungal spores. Ziehl-Neelsen (hot), Kinyoun (cold) are still widely used methods to detect acid-fast structures in these organisms in developing countries but sensitivity is high of fluorochrome stain. The acid fastness of Mycobacterium tuberculosis is due to having a thick cell wall composed of waxes and lipids that has a high content of mycolic acid.

Principle of Auramine -rhodamine stain

Auramine-rhodamine is the fluorochrome dye that forms a complex with mycolic acids found in the acid-fast cell wall of organisms that resist decolorization by acid-alcohol. Potassium permanganate, counterstain renders tissue and its debris non-fluorescent, therefore reducing the possibility of artifacts. The cellular structures visualized under U-V appear bright yellow or reddish-orange.

Test requirements

Auramine Rhodamine stain (Primary Stain)
0.5% Acid alcohol ( Decolorizer)
0.5% Potassium Permanganate
Test specimen (e.g. sputum )
Slide ( clean and grease-free)
Pencil ( diamond if possible)
Slide racks
Bunsen burner
Inoculating loop or sterile bamboo stick

Staining procedure of Auramine -rhodamine

Smear preparation

Take a purulent part of the sputum to a slide and make a thin smear using an inoculating loop or bamboo stick. Cover an area of approximately 2 cm square and spread the smear using circular movements. Allow it to air dry. Finally, perform heat-fixing passing the dried slide, smear facing upward, 2 to 3 times through the blue cone of a burner flame.

Staining

Put the fixed smear on a staining rack and flood the smear with rhodamine-auramine for 15 minutes. Do not let smear dry.
Wash off the stain with clean water.
Decolorize the smear by covering it with acid-alcohol for 2-3 minutes. ( But here in this case of Nocardia, we used 0.025 % acid alcohol i.e. modified form of auramine -rhodamine stain.)
Wash off the acid alcohol with clean water.
Now cover the smear with potassium permanganate for 3-4 minutes. Do not allow smear to dry.
Rinse thoroughly with distilled water and air dry.
Examine the smear by fluorescence microscope and use the 10X objective to focus the smear. Finally, observe the smear using the 40 X objective for acid-fast structures or acid-fast bacilli (AFB).

Result Interpretation

Test Positive: Acid-fast organisms fluoresce bright yellow or reddish-orange against a dark background.
Negative Test: Non-acid-fast organisms will not fluoresce

Precautions

Take precautions during handling the following reagents due to the following reasons-

The auramine-rhodamine stain is a possible carcinogen.
Acid alcohol is flammable and Corrosive.
Potassium Permanganate is also corrosive.

Advantages over Z-N stain

Nearly 10% more sensitive than Z-N stain
It does not require the use of oil immersion fields and thus no need for cedarwood oil.
Heat is not required for auramine-rhodamine staining.

Key Notes

Auramine and rhodamine allow rapid screening of specimens and are used by laboratories that routinely perform acid-fast examinations.
Fluorochrome-stained smears can be restained by the Kinyoun or Ziehl-Neelsen methods.

3. Calcofluor White Stain

Calcofluor white is a non-specific fluorochrome that binds to cellulose and chitin in the cell walls of fungi, including yeast cells, hyphae, pseudohyphae, and spherules. Because of the rapidity and specificity with which specimens can be observed, the stain has become commonplace in microbiology laboratories. The dye can be mixed with 10% potassium hydroxide so that mammalian cells can be dissolved, thus facilitating the visualization of fungal elements. Fungi, Pneumocystis spp., and Acanthamoeba spp. appear green or blue against a dark background when the stained slide is examined under UV illumination. Care must be used to distinguish specific staining from stained debris.

4. Cryptosporidium Stain

Cryptosporidium Stain is used to stain smears made from clinical specimens for the purpose of detecting the oocysts of Cryptosporidium spp. The test employs the use of the dimethyl sulfoxide (DMSO) acid-fast staining procedure. Of the various acid-fast staining procedures, it is the easiest and quickest to perform and provides excellent results. DMSO, a wetting agent with tremendous penetrating qualities, is mixed with carbol fuchsin stain. DMSO transports the carbol fuchsin through the oocyst wall and rapidly stains the red cell, eliminating the need to heat the slide for stain penetration. A mild acetic acid is added to the malachite green counterstain solution combining the decolorization and counterstain steps into one. Oocysts stain red while most other fecal material appears green. Other structures that may stain red can be easily distinguished from oocysts.

5. Direct Fluorescent-Antibody Stain

Specific fluorescein-labeled antibodies are used to directly detect a variety of organisms (e.g., Streptococcus pyogenes, Bordetella pertussis, Francisella tularensis, Legionella spp., Chlamydia trachomatis, Cryptosporidium parvum, Giardia lamblia, influenza virus, herpes simplex virus) in clinical specimens. Specimens are fixed to a glass slide and incubated with the fluorescein-labeled antibody. The labeled antibodies bind to the organisms and fluoresce green under UV light. The sensitivity and specificity of the stain are determined by the quality of the antibodies used in the reagents.

6. Dobell & O’Connor Iodine

A weak iodine solution such as Dobell and O’Connor (diluted Lugol’s) is recommended for use in staining protozoan cysts. This preparation is a 1:5 dilution of Lugol’s Iodine. Iodine Solution is often used in wet mounts of concentrated fecal material. It is useful for staining glycogen and making nuclei visible in protozoan cysts. Protozoan cysts correctly stained with iodine contain yellow-gold cytoplasm, brown glycogen material, and paler refractile nuclei.

7. Flagella Stain

Hardy Diagnostics Flagella Stain is a simple, rapid, qualitative method for detecting bacterial flagella and their shape, length, curvature, arrangement, and number on the cell. This method was developed by Ryu in 1937, and was also later described by Kodaka, et al. in 1982. This test has been found especially useful in providing taxonomic and identifying information about motile bacteria, and more recently, anaerobic bacteria. The Flagella Stain provides a method for viewing bacterial flagella by employing crystal violet in an alcoholic solution as the primary stain. The Flagella Stain also contains tannic acid and aluminum potassium phosphate as mordants, and phenol as an antifungal agent.

8. Giemsa Stain

Introduction of Giemsa stain

Giemsa stain comes under a type of Romanowsky stain. The name of this stain has come from the surname of a German chemist Gustav Giemsa, who created a dye solution. It was initially designed for the detection of malarial parasites in blood smears, but it is also used in histology for routine examination of blood smears. This technique uses for the demonstration of other than malarial parasites, microorganisms like Helicobacter pylori,

Chlamydia trachomatis, Borrelia species, Histoplasma capsulatum, Pneumocystis jiroveci, Penicillium marneffei and occasionally bacterial capsules and parasites like Toxoplasma gondii, Leishmiania donovani , Giardia lamblia, etc. It is also applied to differentiate nuclear and cytoplasmic morphology of the various blood cells like RBCs, WBCs, and platelets.
In cytogenetics, it stains the chromosomes and identifies chromosomal aberrations.

a) Preparation of Giemsa stain

Giemsa stock powder : 1 gm
Glycerin : 54 ml
Methanol : 84 ml

Giemsa powder is mixed in 54 ml of glycerin and pre-heated up to 60°C.

Then add methanol, shake the mixture and allow to stand for 7 days.

Filter before use.

b) Buffer solution (stock)

Potassium dihydrogen phosphate: 2.72 gm
Distilled water: 100 ml
Sodium hydroxide: 0.8 gm
Distilled water: 100ml

Dissolve both powders in distilled water.

50 ml of potassium dihydrogen phosphate is mixed with 23.6 ml of sodium hydroxide.

The pH of the solution is adjusted to 6.8.

Working Giemsa Stain Solutions

Giemsa stock: 10 ml
Working buffer: 90 ml

Should be prepared fresh then use.

Buffer solution

Stock buffer: 20 ml
Distilled water: 480 ml

Principle of Giemsa stain

Giemsa contains Methylene blue(AzureII)/Eosin. Methylene blue on oxidation produces colored compounds termed ‘Azure’ that have the ability to combine with Eosin. Methylene blue azure is blue-violet and stains acidic cell components while eosin is red and stains basic cell components.

Procedure of Giemsa stain

For bone marrow imprints and smears
Smears are fixed in methanol for 30 minutes.

Smears are stained in working Giemsa solution for 20 minutes

Wash under running tap water for 5 minutes.

Air dry smears, clear in xylene and mount in D.P.X.

For Paraffin Section
De-paraffinize and hydrated sections to tap water.

Flood slide with Giemsa stain for 15-20 minutes.

Wash in tap water.

Differentiate 0.2% acetic acid 1 dip.

Wash in running tap water.

Dehydrate, clear and mount with D.P.X

Result interpretation of Giemsa stain

Nuclei: Blue

Cytoplasm: Pink

H. pylori and L.D bodies: Blue

Mast cell: Magenta pink

Tissue elements: Shades of blue to pink

Collagen, Muscle, and Bone: Pale pink

Erythrocytes: Salmon pink

Malaria parasite: Malaria parasites have a red or pink nucleus and blue cytoplasm

Borrelia spirochetes: Mauve-purple

Chlymadia trachomatis inclusion bodies: Blue-mauve to dark purple depending on the stage of development

9. Gram Stain

Introduction of Gram Stain

Gram stain is a differential stain and therefore it uses to differentiate Gram-positive and Gram-negative bacteria. It was devised originally by a Danish bacteriologist, Hans Christian Joachim Gram (1884) as a method of staining bacteria in his laboratory.

Principle of Gram stain

The reaction is dependent on the permeability of the bacterial cell wall and cytoplasmic membrane, to the dye–iodine complex. In Gram-positive bacteria, the crystal violet dye iodine complex combines to form a larger molecule which precipitates within the cell. The alcohol /acetone mixture which acts as a decolorizing agent causes dehydration of the multi-layered peptidoglycan of the cell wall. This causes a decrease in the space between the molecules causing the cell wall to trap the crystal violet iodine complex within the cell. Hence the Gram-positive bacteria do not get decolorized and retain primary dye appearing violet.

Also, Gram-positive bacteria have more acidic protoplasm and hence bind to the basic dye more firmly. In the case of Gram-negative bacteria, the alcohol, being a lipid solvent, dissolves the outer lipopolysaccharide membrane of the cell wall and also damages the cytoplasmic membrane to which the peptidoglycan attaches. As a result, the dye-iodine complex does not retain within the cell and permeates out of it during the process of decolonization. Hence, when a counterstain uses, they take up the color of the stain and appear pink.

Requirements for Gram stain

a) Compound light microscope

b) Reagents and glasswares

Bunsen flame
Wire loop
Clean grease-free slides
Marker pen
Crystal violet (Basic dye)
Gram’s iodine(mordant)
95% ethanol (decolorizing agent)
1% safranin or dilute carbol fuchsin or neutral red

c) Quality control strains

Positive Control (PC) : Staphylococcus aureus (ATCC 25923)

Negative Control (NC): Escherichia coli (ATCC 25922)

d) Specimen

Preparation of bacterial smear: from liquid culture

Take a clean, and grease-free slide for making a smear.
Take one or two loopful of the bacterial cell suspension and place them on the slide with a bacteriological loop.
Then with a circular movement of the loop, spread the cell suspension into a thin area.
Allow the smear to air dry.
Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.

Preparation of bacterial smear: from the solid medium

Take a clean, and grease-free slide for making a smear.
Take a loopful of 0.85% saline i. e. physiological saline and place it on the Center of the slide.
With a straight wire touch the surface of a well-isolated colony from the solid media and emulsify in the saline drop forming a thin film.
Allow the smear to air dry.
Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.

Procedure of Gram Stain

Cover the smear with crystal violet and allow it to stand for one minute.
Rinse the smear gently under tap water.
Cover the smear with Gram’s iodine and allow it to stand for one minute.
Rinse smear again gently under tap water.
Decolorize the smear with 95% alcohol.
Rinse the smear again gently under tap water.
Cover the smear again gently with safranin for one minute.
Rinse the smear again gently under tap water and air dry it.
Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.

Observation of Gram Stain

Positive Control: violet color, round in shape in single, pairs and cluster

Test: red color and rod in shape

Negative Control: red in color and rod in shape

Result and Interpretation of Gram Stain

Gram-positive: purple or violet color

Gram-negative: Pink or red in color

Cocci: round in shape

Bacilli: rod in shape

Positive Control(PC): Gram-positive cocci in single, pairs and cluster

Test: Gram-negative bacilli

Negative Control(NC): Gram-negative bacilli as shown above image.

E. coli under microscope|| Gram stain ||Gram Negative bacilli or Gram-negative rods as shown below-

Variety of bacteria under the microscope showing
Gram-positive bacteria, gram-positive cocci in singles, pairs, clusters i.e. Staphylococcus aureus
Gram-positive rods or bacilli
Bacillus species
Gram-negative bacilli or rods
Salmonella Typhi
Diphtheroids
Sputum gram stained smear
having ideal smear
Gram-positive cocci in pairs inside the pus cell
Gram-positive cocci in chains

A patient was 53 years old with Chronic Otitis Media (COM) having a failure of antibacterial drugs-
Pus swab from ear discharge sent to microbiology section for Gram staining- result found – Fungal spores with plenty of pus cells and lacking bacteria as shown in the video.

Strongyloides stercoralis under Gram stained slide of sputum- a very very rare case-

Impression for this a rare case report from Gram stain of sputum While reporting gram stained slide, we always remember the nature of specimens and availability of organisms; and broaden our vision for report variety of causative agents without missing them because as you know laboratory diagnosis is the third eye of clinicians that makes treatment easier as well as short hospital stay and reduce patient economic burden.

Growth of Candida albicans on SDA and its Gram-stained smear under the microscope and germ tube test (GTT) positive as shown below-

10. India Ink Stain (Nigrosin)

The use of India Ink is not technically a staining method. The polysaccharide capsule of encapsulated organisms (e.g., Cryptococcus neoformans) excludes the ink particles. Organisms are detected by the appearance of a distinctive halo surrounding the encapsulated fungi. Care in interpretation is required because artifacts (e.g., leukocytes, erythrocytes, powder, bubbles) may be confused with yeast cells. The morphological characteristics of the yeast cells must be recognized before the preparation can be interpreted.

India ink or nigrosin preparation (negative staining) for Fungal Infections Laboratory Diagnosis

India ink or nigrosin preparation may be used to examine CSF for the presence of the encapsulated yeast such as Cryptococcus neoformans. With negative stain, budding yeast surrounded by a large clear area against a black background is presumptive evidence of C. neoformans as shown below.

Test Procedure

In CSF for Cryptococcus neoformans:

First centrifuge the CSF.
Take a drop of sediment part on a clean and grease-free slide and add over it a drop of nigrosin.
Mix it properly and put a coverslip over it.
Observe under a microscope, focusing at 10X objective and finally 40X objective.

In the case of bacteria capsule

Take a clean and grease-free slide. Put a drop of nigrosin over it. Touch the colony with inoculating loop from a solid medium or take a loopful broth in case of a liquid medium. Mix the nigrosin drop and make a smear. Leave for air dry. Focus at 10X objective and finally observe at oi immersion lens i.e. 100X objective.

Principle And Interpretation

Negative staining permits visualization of the usually transparent and unstainable capsule of many organisms, most importantly Cryptococcus neoformans. Nigrosin consists of a suspension of fine particles of carbon. These form a dark background, against which capsules see clearly as a result of displacement of the carbon particles.

Quality Control

Appearance blackish violet-colored solution.

Clarity

Clear without any particles.

Results

It is clear halos surrounding the cells as shown above image.

11. Iron Hematoxylin Stain

Iron Hematoxylin Stain is used for the detection and identification of fecal protozoa. Helminth eggs and larvae generally retain too much stain and are more easily identified with wet-mount preparations. Iron Hematoxylin Stain can be applied to either fresh stool specimens or ones preserved with polyvinyl alcohol or a similar preservative. Formalin-fixed specimens cannot be used. Background material and organisms stain gray-blue to black, with cellular inclusions and nuclei appearing darker than the cytoplasm.

12. Kinyoun Stain for AFB

The presence of long-chain fatty acids (e.g., mycolic acid) in some organisms makes these organisms both difficult to stain with water-soluble dyes and resistant to decolorization with acid solutions (i.e., the organisms are considered acid-fast). The Kinyoun method of staining uses high concentrations of basic carbol fuchsin and phenol to facilitate penetration of the dye into the cells. The increased concentration of these two components avoids the need for heating the slide and is therefore referred to as a cold acid-fast stain. Basic carbol fuchsin is used as the primary stain, 3% sulfuric acid in 95% ethanol (acid-alcohol) is the decolorizing agent, and methylene blue is the counterstain. Acid-fast organisms appear pink-red on a pale blue background. The contrast between organisms and background is sometimes poor, and fluorochrome stain is generally preferred for specimen examination. Acid-fast stains are used for detecting bacteria, including Mycobacterium, Nocardia, Rhodococcus, Tsukamurella, and Gordona spp., and the oocysts of Cryptosporidium spp., Isospora belli, Sarcocystis spp., and Cyclospora spp. Because some of these organisms lose the primary stain when they are exposed to 3% sulfuric acid, the decolorizing agent can be reduced to 0.5 to 1%. Organisms that retain this modified stain are referred to as being partially acid-fast.

13. Lactophenol Cotton Blue

Introduction of LPCB stain

LPCB stain stands for lactophenol cotton blue and it is a combination of fixative, staining, and clearing agent. LPCB uses both as a mounting fluid and a stain. This is used for staining and microscopic identification of fungi. Its contents functions are as follows-

Lactic acid: It helps in preserving the morphology of the fungal elements.

Phenol: It acts as a disinfectant.

Cotton blue: It 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).

Glycerol: It is a hygroscopic agent that prevents drying.

Principle of LPCB stain

Ingredients of LPCB stain like lactic acid acts as a clearing agent and aids 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 such as shape, size, arrangement of spores, and hyphae providing color to the structure. It can be used alone or in conjunction with KOH.

Composition of LPCB stain

For 50 ml
Lactic acid : 10 ml
Phenol : 10 ml
Glycerol :20 ml
Cotton blue (Poirier blue or Aniline blue): 0.025 g
Distilled water : 10 ml

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

Requirements for test

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

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 of air bubbles.
Examine under low- (10 X) and high-power (40 X) objectives.
Observe the morphological features carefully as shown below.
Observation
Fungi appear as dark blue stained mycelium.
Results and interpretations
Different fungi under LPCB wet mount will show different types of morphological structures including hyphae and spores. We concern with Aspergillus as shown below.
Fungal spores, hyphae, and fruiting structures: Takes stain blue
Background: stains pale blue.
Application of 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 Cyst of intestinal protozoa and ova takes blue color while ova of helminths are stained deep blue.
Various fungi and their structures ( yeast cells, budding yeast, hyphae, pseudohyphae, mycelium, spores) in LPCB preparation are as follows-

Aspergillus fumigatus Colony on SDA, LPCB tease mount under microscopy

Trichosporon on SDA and lactophenol cotton blue preparation under the microscope

Geotrichum growth on SDA and its fungal structures on lactophenol cotton blue preparation

Bipolaris growth on SDA and its structures on lactophenol cotton blue preparation

Syncephalastrum in lactophenol cotton blue preparation under the Microscope

Penicillium colonial morphology and its microscopic features in lactophenol cotton blue tease mount under microscope

Fungus, Acremonium on SDA and lactophenol cotton blue preparation

Aspergilus flavus on Czapek Dox agar, Cornmeal agar, and lactophenol cotton blue tease mount

Fusarium growth on SDA and its structures in lactophenol cotton blue preparation

Cryptococcus neoformans in lactophenol cotton blue tease mount

Candida albicans in LPCB tease mount

Cladosporium on SDA and its fungal structures on lactophenol cotton blue preparation

LPCB Mount of Curvularia species

Mucor in LPCB mount

Lactophenol cotton blue tease mount procedure and observation under the Microscope

Sporothrix schenckii under the microscope in lactophenol cotton blue preparation showing the following structures-conidia, conidiophores, and septate hyphae
Conidia in clusters

Trichophyton mentagrophyte Isolated: features-
Helical pattern on lactophenol cotton blue Mount seen
Urease test_Positive
Hair perforation test-Positive

Arthroconidia of Trichosporon inkin – Long Cylindrical in Shape

Note: For more videos, click this link@https://www.youtube.com/channel/UC_Kvqp1w3_Ey-ifaQBsxfNg

Key Notes

LPCB wet mount is always examined at least 30 minutes after preparation.
A wet mount preparation should neither be too thick or too thin.
In this preparation, both bile-stained and non-bile-stained helminthic eggs are stained blue.
LPCB kills the trophozoites of Entamoeba and Trichomonas, hence, can not be demonstrated by this.
In LPCB wet mount of stool phenol and lactic clear fecal debris.
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 being very useful has some shortcomings like-

It is only applicable to the presumptive identification method of fungi.
The ingredient of the LPCB solution may disrupt the original morphology of the fungi.
The stain can only be used to identify mature fungi and their structures and not the young vegetative forms of fungi.
A wet mount preparation should neither be too thick or too thin.
Application of LPCB stain in Parasitology is not preferred because it kills the trophozoites of Entamoeba and Trichomonas.
This LPCB stain has an expiry date and thus can only use before expiry.

14. Lugol’s Iodine Stain

Iodine, a non-specific contrast dye, is added to wet preparations of parasitology specimens to enhance the contrast of the internal structures (e.g., nuclei, glycogen vacuoles). Parasitic cysts take up the dye and appear light brown, and are thereby differentiated from WBCs. One disadvantage of this method is that protozoa are killed by the iodine and thus motility cannot be observed.

15. Methenamine Silver Stain

Methenamine silver staining is generally performed in surgical pathology laboratories rather than microbiology laboratories. It is primarily used for the detection of fungal elements in tissues, although other organisms (e.g., Legionella spp.) can be detected. Silver staining requires skill because non-specific staining can render the slide uninterpretable. Fungi are delineated in black against a pale green background. Inner parts of hyphae appear charcoal gray.

16. Methylene Blue Stain

Methylene blue is another contrasting dye commonly used in the laboratory, primarily for the detection of bacteria and fungi. It can be mixed with potassium hydroxide and used to examine skin scrapings for fungal elements. Methylene blue is also used for detecting fecal WBCs. The presence of WBCs in the stool is suggestive of invasive bowel disease.

17. Periodic Acid-Schiff (PAS) Stain

Periodic Acid-Schiff Stain is used to detect yeast cells and fungal hyphae in tissues. Periodic acid (5%) hydrolyzes the cell wall aldehydes, which then combine with the modified Schiff reagent and stain the cell wall carbohydrates pink-magenta against a light green background. The stain is time-consuming and also requires the specimens to be digested. Because this staining procedure is complex, most laboratories have replaced it with the calcofluor white stain.

Introduction of Periodic acid Schiff’s (PAS) Stain

PAS stain is used for the demonstration of basal membrane, fungus, differentiate mucin secreting adenocarcinoma from undifferentiated squamous cell carcinoma. It is helpful in the diagnosis of different types of glycogen storage diseases, Paget’disease, macrophages in Whipples diseases, parasites, and amylase.

Principle of PAS stain

Structures having 1-2 glycol or amino or alkyl grouping are oxidized by periodic acid. Free hydroxyl groups must be present for oxidation. After complete oxidation, a dialdehyde is formed which is colorless and unstable. This aldehyde after treating with Schiff’s reagent turned to a magenta red-colored product.

Test requirements

Reagent
Hematoxylin
Periodic acid
Hydrochloric acid (HCl)
Potassium
Metabisulphite
Alcohol
Activated charcoal
Basic fuchsin
supplies
Gloves
Masks
Lab coat/ apron
Cotton
Equipment
Coplin jar
Measuring cylinder
Conical flasks
Glassware
Reagents bottles

Preparation and Procedure of PAS stain

Preparation of reagents (0.5% Periodic acid)

Periodic acid: 0.5 gm
Distilled water: 100 ml

Preparation of Schiff reagent

basic fuchsin – 1 gm
distilled water – 200 ml
potassium meta bisulfate – 2 gm
Concentrated HCl – 2 ml
activated charcoal – 2 gm

Dissolve 1 gm of basic fuchsin in 200 ml of boiling distilled water by removing the flask of water from the burner just before adding the basic fuchsin.
Allow the solution to cool at 50°C.
Add 2 gm of potassium metabisulphite and mix properly. Allow cooling to room temperature.
Add 2 ml concentrated hydrochloric acid and mix.
Leave overnight in the darkroom temperature and add 2 gm activated charcoal. Filter through a no. 1 Whatman filter paper, the solution should be
colorless.
Store in an amber-colored container at 4°C.

Preparation of Harris’s Hematoxylin ( 500ml )

Hematoxylin – 2.5 g
Absolute alcohol – 25 ml
Potassium alum – 50 gm
Distilled water – 500 ml
Mercuric oxide – 1.25 g
Glacial acetic acid – 20 ml

Dissolved Hematoxylin in absolute alcohol
Take hot distilled water and mix alum until dissolved well
Add mercuric oxide carefully and cool in cold water, taking care of
excess bubbling.
Add glacial acetic acid

Procedure of PAS stain

Deparaffinize and hydrate to distilled water.
Place slide into 0.5% periodic acid for 5-7 minutes.
Wash with several changes of distilled water.
Cover with Schiff’s solution for 20 minutes until dip magenta color is seen.
Then wash in running tap water.
Counterstain with Harris hematoxylin for 1 minute.
Wash in running tap water.
Dehydrate in alcohol, clear in xylene, and mount in D.P.X.

Result interpretation of PAS stain

Nuclei: Blue
Glycogen, fungus: Magenta/ deep pink
Diastase treated tissues: Colorless

Note: Fungi stain a bright pink-magenta or purple against a green background
when light green is used as a counterstain.

Precautions for mycological aspects

A slide of either skin or nail scrapings containing a dermatophyte should be stained along with slides of the specimen as a positive control. Periodic acid may deteriorate and no longer oxidize the hydroxyl groups. This should be suspected when fungal elements on the control slide appear unstained. The
periodic acid solution and the stock of periodic acid (a white powder)
should be kept in dark bottles. The sodium metabisulphite solution is unstable. Deterioration of this reagent is suspected when the control slides
show no evidence of having been subjected to a bleaching process, e.g. the
background stains as intensely as they do the fungal elements.

18. Sudan Black B Stain

Sudan Black B Stain is a qualitative method for detecting the presence of fat granules in Bacillus species and other bacteria. This procedure, when performed carefully and consistently, aids in the detection of poly-beta-hydroxybutyrate granules in bacteria. Sudan Black B Stain was originally prepared in Germany in the early 1930s. Lison and Dagnelie proposed its use as a myelin stain in 1935. Gerard, also in 1935, first developed its use as a fat stain. In the 1940s, both Hartman and Burdon further established their value as a bacterial fat stain. It has also been proposed for staining Golgi apparatus and leukocyte granules.

19. Sudan Black IV Stain

Sudan Black IV Stain is used as a qualitative method to detect the presence of fecal fat. In the case of steatorrhea, fat malabsorption occurs, and high quantities of fat are detected in the stool. This procedure, when performed carefully and consistently, is a simple method of detecting this condition in the patient. Sudan Black IV Stain, also known as scarlet red, was introduced by Michaelis in 1901 as a fat stain. It is a dimethyl derivative of Sudan III, which makes it a deeper and more intense stain, yet it has similar physical properties and is fat-soluble. This stain has been widely used as a screening method because it is easy to use, and correlates well with quantitative methods.

20. Trichrome Stain

Trichrome Stain, like Iron Hematoxylin Stain, is used for the detection and identification of protozoa. Trichrome Stain produces well-stained smears from both fresh and PVA-preserved material. When staining is done properly, the specimen background is green and the protozoa have blue-green to purple cytoplasms with red or purple-red nuclei and inclusions.

Importance of Wheatley’s Modification of the Gomori Trichrome stain

Identification of cysts and trophozoites in fecal specimens is very useful for the diagnosis of intestinal parasitic infection conformation. Since smaller protozoans often go undetected in direct wet mount and concentration methods, the identification of intestinal protozoa depends on the examination of a permanent stained smear. Wheatley’s Modification of the Gomori Trichrome stain provides excellent detail and contrast with preserved specimens. For example trophozoite of Giardia as shown above image

Principle of Trichome stain

Chromotrope 2R has an affinity for chromatin material. Nuclear chromatin, karyosomes, chromatoid bodies, parasite eggs and larvae,
bacteria, and ingested erythrocytes stain red to purple-red. Light green and fast green dyes stain the cytoplasm of preserved cysts,
trophozoites, and cellular constituents blue-green. The Trichrome
Stain results in excellent contrast and visualization of cellular details that help in the identification of protozoa.

Requirements

Wheatley Trichrome Stain: It contains the following ingredients and they are-
Phosphotungstic Acid: 7.0 g
Chromotrope 2R: 6.0 g
Light Green SF: 1.5 g
Fast Green FCF: 1.5 g
Glacial Acetic Acid: 10.0 ml
Demineralized Water: 1000.0 ml
Xylene
Ethanol 95%
Ethanol 70%
Acid Ethanol 90%:
Lugol’s Iodine Ampules
Permount
Coverslip
Clean and grease-free slides
Warmer
Gloves
Times
Applicator stick or sterile bamboo stick
Absorbent paper( paper towels)
Coplin jars
staining rack
forceps
Pipettes
Microscope
Immersion oil (cedarwood oil)

Quality Control

A control slide of a known protozoan parasite e.g. Giardia species from a Polyvinyl alcohol (PVA) preserved specimen should be included with each staining run.

Procedure of Wheatley’s Modification of the Gomori Trichrome stain

Smear preparation

Wear gloves when performing this procedure.
Allow stool specimens preserved in polyvinyl alcohol to fix at least 30 minutes whereas mix fresh specimens received in the laboratory with PVA (1 part feces to 3 parts fixative) and allow fixing for 30 minutes.
Thoroughly mix the specimen and the PVA. Pour a small amount of the mixture onto a paper towel to absorb excess fixative. Allow the fixative to soak into the paper towel for 3 minutes before preparing slides.
With an applicator stick transfer some of the stool material from the paper towel to 2 clean glass slides. Spread the mixture to the edges of the slide so the specimen will adhere to the slide during staining. Note: The amount of material applied to the slide should be thin enough that newsprint can be read through the smear.
Allow the slides to dry overnight at room temperature or 60ºC for 4 minutes in warmer or an hour at 35-37ºC.

Staining

Immerse slides prepared from fresh specimens in ethanol-Iodine for 1 minute, whereas immerse PVA-preserved, air-dried smears in ethanol-Iodine for 5-10 minutes.
Place slides in 70% ethanol for 5 minutes. Drain excess liquid.
Place slides in a second jar of 70% ethanol for 3 minutes.
Place slides in Wheatley Trichrome stain for 10 minutes.
Place slides in acid ethanol for 1-3 seconds. Immediately proceed to the next step. Do not allow the slides to remain in contact with this solution longer than 3 seconds.
Dip slides several times in 95% ethanol.
Place slides in two changes of 95% ethanol for 3 minutes each.
Place slides in two changes of Xylene for 5-10 minutes each.
Apply mounting medium (e.g. permount) to the smear and cover with a No. 1
thickness cover-slip.
Allow the smear to dry overnight at room temperature or for 1 hour at 35-37°C.
Examine the slide microscopically, using the oil immersion objective for nuclear detail. At least 200-300 oil immersion fields
should be examined.

Result Interpretation of Wheatley’s Modification of the Gomori Trichrome stain

Fixatives are responsible for variation in staining characteristics and the typical staining reactions with Trichrome Stain are as follows:

Nuclear chromatin, chromatoid bodies, ingested erythrocytes,
and bacteria: They stain red to purple-red.
Cytoplasm: It stains blue-green with a faint purple tinge.
Macrophages, leukocytes, and yeast cells: They vary in staining reactions.
Background material: It stains green providing a nice color contrast with the protozoa/
Glycogen: It is dissolved by the solvents and appears as a clear area in the organism.

20. Uristain

The stain contains various dyes that aid in differentiating the abnormal and normal cellular elements found in urine. UriStain™ is a one solution modification of the Sternheimer and Malvin procedure. This combination stain includes Ammonium Oxalate, Safranin, Crystal Violet, and Ethanol.

21. Wright Stain

Wright Stain is used in the differential staining of basophilic and acidophilic material. It is a polychromatic stain that contains a mixture of methylene blue, azure B (from the oxidation of methylene blue), and eosin Y dissolved in methanol. The eosin ions are negatively charged and stain the basic components of the cells orange to pink, while the other dyes stain the acidic cell structures various shades of blue to purple. Like Giemsa Stain, it is primarily used for the differentiation of intracellular and extracellular circulating blood parasites (e.g., Plasmodium, Babesia, and Leishmania spp.), fungi (e.g., Histoplasma spp., yeast cells, Pneumocystis spp.), rickettsiae, chlamydiae, and viral inclusions.

22. Ziehl-Neelsen Stain for AFB

Ziehl-Neelsen, an acid-fast stain, requires that the specimen be heated during staining so that the basic carbol fuchsin can penetrate into the organisms. Once the dye is forced into the cell, decolorization and counterstaining are the same as with the Kinyoun method. The sensitivity and specificity of this stain are essentially the same as those of the Kinyoun method. The Kinyoun Stain procedure, however, is less time-consuming and easier to perform.

Introduction of AFB staining

This acid-fast bacilli in brief AFB staining or Ziehl-Neelsen staining method is a modification of Ehrlich’s (1882) method. Its name is from the surnames of German doctors, bacteriologist Franz Ziehl(1859-1926), and pathologist Friedrich Neelsen (1854-1898).

Principle of AFB Staining

The presence of higher alcohol, glycerol, fatty acid, and especially mycolic acid in the cell wall has been found responsible for keeping the acid-fast property of bacteria. Therefore, AFB staining is useful for Mycobacterium tuberculosis, an etiological agent of tuberculosis.

Requirements

a) Compound light microscope

b) Reagents and glasswares

Bunsen flame
Wire loop
Clean grease-free slides
Marker pen
Sprit lamp
Carbol fuchsin
20% Sulphuric acid
Methylene blue

c) Specimens

In the case of primary tuberculosis

sputum
bronchial or laryngeal washing
Gastric lavage when sputum is swallowed as in children

In miliary tuberculosis

bone marrow
Liver biopsy

Tuberculous meningitis

Cerebrospinal fluid (CSF)

Renal tuberculosis

urine

d) Quality control strains

Positive control (PC): Mycobacterium tuberculosis
Negative Control: Escherichia coli

Procedure of AFB staining

Make smear on a clean glass slide.
Dry and fix the smear.
Cover the smear with a strong carbol fuchsin solution.
The heat from underneath the slide until just steam comes from the stain. Do not boil.
Wait for five minutes.
Rinse with water.
Decolorize by 20% Sulphuric acid or 3% acid alcohol until the smear becomes pale pink in color. (wait for nearly five minutes)
Rinse with water.
Counterstain with methylene blue for one minute.
Rinse with water.
Drain and dry.
Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.

Result and Interpretation

AFB: pink or red bacillus

Background: Blue ( as counterstain used )

Reporting

There are various ways of a reporting system for AFB stainings such as the Center for Disease Control and Prevention (CDC), World Health Organization (WHO), and International Union Against Tuberculosis and Lung Disease (IUATLD). The most common and widely accepted classification is IUATLD and according to it as follows.

No organism is seen: Negative
1-9/100 OIF ( oil immersion field): Exact number
10-99/100 OIF: +
1-10/OIF : ++
10/OIF: +++

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