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Microorganisms in Tissues 2 2021

The document discusses fungi that can infect tissues. It describes common superficial fungal infections of the skin and outlines staining techniques used to identify fungi in tissue like Grocott's methenamine silver stain and PAS stain. It also lists some more important fungal and bacterial infections.

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Karungi immaculate
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24 views16 pages

Microorganisms in Tissues 2 2021

The document discusses fungi that can infect tissues. It describes common superficial fungal infections of the skin and outlines staining techniques used to identify fungi in tissue like Grocott's methenamine silver stain and PAS stain. It also lists some more important fungal and bacterial infections.

Uploaded by

Karungi immaculate
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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MICROORGANISMS IN

TISSUES

26/07/2021 FRANK S; MMLS 1


FUNGI

26/07/2021 FRANK S; MMLS 2


FUNGI
• Fungi are widespread in nature, and humans are
regularly exposed to the spores from many species.

• The most commonly encountered diseases are the


superficial mycoses that affect the subcutaneous or
horny layers of the skin or hair shafts, and cause
conditions such as athlete’s foot or ringworm.

• These dermatophytic fungi belong to the Microsporum


and Trichophyton groups and may appear as yeasts or
mycelial forms within the keratin.
26/07/2021 FRANK S; MMLS 3
FUNGI
• They are seen fairly well in the H&E stain, but
are demonstrated well with the Grocott and PAS
stains.

• As with other infections, the increase in the


number of patients with diminished or
compromised immune systems has increased the
incidence of systemic mycoses, allowing
opportunistic attacks by fungi, often of low
virulence, but sometimes resulting in death.
26/07/2021 FRANK S; MMLS 4
FUNGI
• When fungi grow in tissue they may display primitive
asexual (imperfect) forms that appear as either spherical
yeast or spore forms.

• may produce vegetative growth that appears as tubular


hyphae that may be septate and branching; these features
are important morphologically for identifying different
types of fungi.

• A mass of interwoven hyphae is called a fungal mycelium.


Only rarely, when the fungus reaches an open cavity, the
body surface, or a luminal surface such as the bronchus, are
the spore-forming fruiting bodies called sporangia, or
conidia, produced.

26/07/2021 FRANK S; MMLS 5


Identification of fungi
• Some fungi may elicit a range of host reactions
from exudative, necrotizing, to granulomatous;
other fungi produce little cellular response to
indicate their presence.

• Fortunately, most fungi are relatively large and


their cell walls are rich in polysaccharides, which
can be converted by oxidation to dialdehydes and
thus detected with Schiff’s reagent or hexamine
silver solutions.
26/07/2021 FRANK S; MMLS 6
Identification of fungi
• Fungi are often weakly hematoxyphilic and
can be suspected on H&E stains.

• Some fungi, such as sporothrix, may be


surrounded by a stellate, strongly eosinophilic,
refractile Splendore-Hoeppli precipitates of
host immunoglobulin and degraded
eosinophils.

26/07/2021 FRANK S; MMLS 7


Identification of fungi
• Fluorochrome-labeled specific antibodies to
many fungi are available, and can be used in
mycology laboratories for the identification of
fungi on fresh and paraffin sections.

• These antibodies have not found widespread


use, however, on fixed tissue where
identification still relies primarily on
traditional staining methods.

26/07/2021 FRANK S; MMLS 8


Identification of fungi
Grocott methenamine (hexamine)-silver for fungi
Sections
• Formalin fixed, paraffin.
Solutions
• 4% chromic acid, commercially available
• 1% sodium bisulfite
• 5% sodium thiosulfate
• (A) 0.21% silver nitrate (stock)
• (B) Methenamine-sodium borate solution (stock)
• Methenamine-silver sodium borate solution (working) Equal parts
of solutions A and B. Make fresh each time and filter before use.
• 0.2% light green (stock)
• Light green (working)
• Prepare working solution fresh before each use.

26/07/2021 FRANK S; MMLS 9


Identification of fungi
Grocott methenamine (hexamine)-silver for fungi
Method
1. Deparaffinize and rehydrate through graded alcohols to distilled water.
2. Oxidize in 4% aqueous chromic acid (chromium trioxide), 30 minutes.
3. Wash briefly in distilled water.
4. Dip briefly in 1% sodium bisulfite.
5. Wash well in distilled water
6. Place in preheated (56–60°C water bath) working silver solution for 15–20 minutes.
Check control after 15 minutes. If section is ‘paper bag brown’ then rinse in distilled
water and check under microscope. If it is not ready, dip again in distilled water and
return to silver. Elastin should not be black. Check every 2 minutes from that point
onwards.
7. Rinse well in distilled water.
8. Tone in 0.1% gold chloride, 5 seconds. Rinse in distilled water.
9. Place in 5% sodium thiosulfate, 5 seconds.
10. Rinse well in running tap water.
11. Counterstain in working light green solution until a medium green (usually 5–15
seconds).
12. Dehydrate, clear, and mount.

26/07/2021 FRANK S; MMLS 10


Identification of fungi
Grocott methenamine (hexamine)-silver for fungi
Results
• Fungi, pneumocystis, melanin ………. black

• Hyphae and yeast-form cells ………. sharply


delineated in black of fungi

• Mucins and glycogen …….. dark gray/ brown

• Background ………………..… pale green


26/07/2021 FRANK S; MMLS 11
Identification of fungi

• Read and make notes on the McManus’ PAS


method for glycogen and fungal cell walls

26/07/2021 FRANK S; MMLS 12


Identification of fungi
A strong hematoxylin shows the fine detail of many infectious agents. The hyphal
structure identifies this as Aspergillus which was colonizing an old
tuberculosis cavity in the lung.

26/07/2021 FRANK S; MMLS 13


Identification of fungi
Grocott’s methenamine-silver stains a wide variety of infectious agents. Here seen with light
green counterstain is the method of choice for Histoplasma capsulatum, a dimorphic endemic
fungus.

26/07/2021 FRANK S; MMLS 14


The more important fungi
and actinomycetes
• Actinomyces israelii
• Nocardia asteroides
• Candida albicans
• Aspergillus fumigatus
• Cryptococcus neoformans
• Pneumocystis jiroveci.

26/07/2021 FRANK S; MMLS 15


THE END

26/07/2021 FRANK S; MMLS 16

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