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Essential Microbiology Lab Techniques

This document outlines procedures for several laboratory tests including: 1. Gram stain - used to classify bacteria as gram positive or negative based on cell wall structure and staining properties. 2. Acid-fast bacilli (AFB) stain - used to identify Mycobacteria by staining their lipid-rich cell walls. 3. KOH preparation - used to visualize fungal elements by clearing tissues in specimens using potassium hydroxide solution. 4. Tzanck smear - examines scrapings from vesicle bases for herpes virus or other infections by staining cells. 5. Malaria smear - examines blood films for malaria parasites after staining. Detailed steps and examples are provided

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Jinnasit Tee
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111 views44 pages

Essential Microbiology Lab Techniques

This document outlines procedures for several laboratory tests including: 1. Gram stain - used to classify bacteria as gram positive or negative based on cell wall structure and staining properties. 2. Acid-fast bacilli (AFB) stain - used to identify Mycobacteria by staining their lipid-rich cell walls. 3. KOH preparation - used to visualize fungal elements by clearing tissues in specimens using potassium hydroxide solution. 4. Tzanck smear - examines scrapings from vesicle bases for herpes virus or other infections by staining cells. 5. Malaria smear - examines blood films for malaria parasites after staining. Detailed steps and examples are provided

Uploaded by

Jinnasit Tee
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd

Laboratory procedure

For medical student


Outline
• Gram stain
• AFB
• KOH preparation
• Tzanck’s smear
• Malaria
Gram stain
Gram stain
• Gram positive bacteria (thick layer of peptidog
lycan-90% of cell wall)- stains purple
• Gram negative bacteria (thin layer of peptidogl
ycan-10% of cell wall and high lipid content) –s
tains red/pink
Equipment
• 10-30% KOH solution
• Light microscope
• Blade
• Coverslip
• Slide
Procedure
• Flood air-dried, heat-fixed smear of cells for 1
minute with crystal violet staining reagent
• Wash slide in a gentle and indirect stream of t
ap water for 2 seconds
• Flood slide with the mordant: Gram’s iodine. 
Wait 1 minute.
• Wash slide in a gentle and indirect stream of t
ap water for 2 seconds.
Procedure
• Flood slide with decolorizing agent (Acetone-alc
ohol decolorizer). Wait 10-15 seconds
• Flood slide with counterstain, safranin. Wait 30 s
econds to 1 minute
• Wash slide in a gentile and indirect stream of tap
water
• Observe the results of the staining procedure un
der oil immersion (100x) using a Bright field micr
oscope.
Procedure
Overview Bacteria
Examples

Gram positive cocci Gram positive cocci


in pair and chain in cluster
Examples

Gram negative intracellular Safety pin-like


diplococci gram negative bacilli
AFB
AFB
• Acid Fast staining technique is used to stain Myc
obacterium species including M. tuberculosis, M
. laprae, NTM
• The cellwall of Mycobacteria contain high conce
ntration of lipid making them waxy, hydrophobi
c, and impermeable to routine stain such as the 
gram stain.
• Ziehl-Neelsen technique (hot staining) Kinyoun t
echnique (cold staining)
Equipment
• Light microscope
• Coverslip
• Slide
• Carbol fuchsin stain
• 1% sulfuric acid
• methylene blue stain
Procedure(Kinyoun)
• Flood slide with Carbol fuchsin, and stain for 5 min.
• Rinse thoroughly with water.
• Decolorize with 1% sulfuric acid for 2 min or until n
o more color runs from the slide.
• Rinse slide with water.
• Counterstain with methylene blue for 1 min.
• Rinse slide with water. Air dry.
• Examine the smear, using the 100x objective 
Examples
KOH preparation
KOH preparation
• KOH preparation is used for the rapid detectio
n of fungal elements in clinical specimen such
as skin, hair, nails or sputum
• When specimen is mixed with 20% KOH, it soft
ens, digests and clears the tissues (e.g., keratin
present in skins) surrounding the fungi so that
the hyphae and conidia (spores) of fungi can b
e seen under microscope.
Equipment
• 10-30% KOH solution
• Light microscope
• Blade
• Coverslip
• Slide
Procedure
• Place 1-2 drops of KOH solution on a slide with t
he specimen, and cover with glass.
• To assist clearing(usually takes 5-15 min), hairs s
hould not be more than 5 mm long, and skin sca
les, crusts and nail snips should not be more tha
n 2 mm across.
• The slide is gently heated to speed up the action
of the KOH
• Examine the slide microscopically using the 10X
and 40X objectives
Examples

Spaghetti & meatball


Branching septate hyphae
(clusters of yeast and short mycelia
filament)
Examples

Pseudohyphae dichotomously branching hyphae


with budding spores
Tzanck’s smear
Tzanck’s smear
• for diagnosis of herpetic infection or bullous d
isease
Equipment
• Wright’s/Giemsa/methylene blue stain
• Light microscope
• Blade
• Coverslip
• Slide
Procedure
• scrape at vesicle base
• air dried
• stain with Wright’s/Giemsa/methylene blue st
ain
Examples

Multinucleated giant cell


Malaria
Procedure
Thank you
ABG Quiz
For medical student
Q1
Q2
Q3

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