Pewarnaan

Sederhana, KOH,
LPCB dan Gram
Pembimbing: dr Rendra Bramanthi, Sp.MK (K)

PPDS MIKROBIOLOGI KLINIK

FAKULTAS KEDOKTERAN
UNIVERSITAS BRAWIJAYA
 Fungal Identification

Sample Handling in Fungal Identification

● Types of sample depends on the pathogenesis of each fungus.
● Possible samples are:

○ Body fluids (cerebrospinal fluid, blood, urine, etc)

○ Respiratory secretion

○ Hair, skin, nail scraping

○ Tissue (including bone marrow)

● Sample must be collected using aseptic method
● To minimize bacterial growth, all samples must be processed within 2 (two) hours after collection.

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 Fungal Identification

Microscopic Identification

Molds Yeast

• look for: • look for:

• hyphae • budding cell
(septate / • pseudohyphae
aseptate) • germ tube
• conidiophore
• conidia
(macroconidia /
microconidia, if
any)
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 Fungal Identification

Microscopic Identification
● Many methods can be used to stain fungi
● Several fungal stain methods are enhanced for particular fungi
● Staining methods i.e:

Colloidal Lactophe Methena

Calcofluo carbon Giemsa nol mine
r white wet stain cotton silver
Periodic
mounts blue stain
acid Hematox
Toluidin
Schiff ylin - etc
blue O
(PAS) Eosin
stain
See notes for detailed explanation 6
LPCB (Lactophenol Cotton Blue)
● The LPCB stain has following three components :

○ Phenol : Kills any live organism.

○ Lactic acid : Preserves fungal structures.

○ Cotton blue : Stains the chitin and cellulose of the fungal cell wall intensely blue

Lactophenol Cotton Blue Solution is a mounting medium and staining agent used in the preparation of slides for microscopic
examination of fungi. Fungal elements are stained intensely blue.
 Fungal Identification

Microscopic Identification
LPCB staining
More preferable for molds

take the
cover the
put one drop specimen make
suspension
of LPCB onto using suspension
with
object glass sterilized with LPCB
coverglass
loop

one loop of
1 drop of sample
LPCB

make suspension but cover with coverglass

do not spread the
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 Fungal Identification

Microscopic Identification
KOH mount

● KOH is basically not ‘stain’, instead it is a strong alkali that readily digests tissues
● KOH digests proteinaceous components surrounding the fungi, leaving the
fungal cell wall intact and more apparent, making the fungi easier to observe
● Digestion is enhanced by heating
● Specimen is prepared as wet mount treated with 10% KOH

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 Microscopic Identification
KOH mount

● Potassium hydroxide (KOH) preparation is used for the rapid detection of fungal elements in clinical specimen, as it clears the
specimen making fungal elements more visible during direct microscopic examination.
● In diagnostic laboratories, KOH mount is one of the main methods of investigating fungal infections. It is used as a primary
screening tool, it detects fungal elements present but may not necessarily identify the species of the fungi.
● It can be used for either yeast and mold, but it does not show molds’ characteristic as clear as staining (i.e. LPCB), so it is more
frequently performed to identify yeasts
 Fungal Identification

Microscopic Identification
KOH mount
More preferable for yeasts

put one take the

make cover the
drop of specimen gently heat
suspension suspension
KOH 10% using the
with KOH with
onto object sterilized specimen
10% coverglass
glass loop

one loop of
One drop of sample
KOH 10%

make suspension but cover with coverglass

do not spread the gentle heat 11
15
GRAM STAIN
PREANALYTICAL CONSIDERATION
PRINCIPLE
● classifies bacteria on the basis of their cell wall structure
● allows observation of their size and cellular morphology as well
● utilized to assess the quality of clinical specimens
● as a critical test for rapid and presumptive diagnosis of infective agents directly from specimens
● Gram-positive species have a thick peptidoglycan layer and large amount of teichoic acids  unaffected by alcohol
decolorization  retain the initial stain  appearing deep violet if their cell walls are undamaged (by age, antimicrobial agents,
etc)
● Gram-negative bacteria have a single peptidoglycan layer attached to an asymmetric lipopolysaccharide-phospholipid bilayer
outer membrane interspersed with proteins  outer membrane is damaged by the alcohol based decolorizer  the crystal violet-
iodine complex leaks out  replaced by the counterstain
SPECIMEN COLLECTION, TRANSPORT, AND HANDLING
SPECIMENS
● Clinical specimens (excluding throat swabs, nasal swabs, sputum from cystic fibrosis patients, fecal material, and prosthetic
devices)
● Direct smears (wounds, eye lesions, sterile fluids, abscesses, and body tissues)
● Broth and positive blood cultures
● Colonies growing on solid media
SPECIMEN COLLECTION
● Smear is made in the laboratory (when transport will be delayed or the preservative for culture will alter the specimen  smears
may be prepared by the collector and the slides transported to the laboratory for staining)
REJECTION CRITERIA
● Little value for direct smears of stool, throat samples, and sputum from cystic fibrosis patients
● Used only occasionally for urine samples
● Not routinely performed on blood cultures due to the low organism density and low yield in most samples
● Not routinely performed on catheter tip specimens
MATERIALS
REAGENTS
• Methanol
• Crystal violet
• Iodine
• Decolorizers
- Slowest: ethanol 95%
- Intermediate: acetone-alcohol (mixed 50:50)
- Fastest: acetone
• Counterstain (safranin)
OTHER SUPPLIES
● Wax pencil
● Precleaned glass slide (25 by 75mm)
● Sterile 0.85% NaCl, water, or broth
● Sterile pasteur pipettes, wood aplicator sticks, inoculating loops, or needles
● Container for biological waste
● Sterile tubes with caps
● Sterile scissors, scalpels, and forceps
EQUIPMENT (DEPENDING ON SPECIMEN SOURCE OR LABORATORY PROTOCOL)
● Benchtop sterilizer (e.g., Bacti-Cinerator or flame burner) with automaticshutoff
● Electric slide warmer, 45 to 60C
● Cytospin centrifuge
● Vortex mixer
● Container or device for collection of toxic stains for chemical hazard disposal
ANALYTICAL CONSIDERATION
PROCEDURE
SLIDE PREPARATION
General consideration
● Place frosted-end glass slides in a container of 95% ethanol
● Using forceps, drain excess alcohol and flame slide prior to use
● Label the frosted ends with the information to identify the specimen or culture
For direct smears
Prepare a monolayer of organisms sufficiently dense for easy visualization but sparse enough to reveal characteristic arrangements
(newspaper print should be visible through the smear)
a. Body fluid, bronchoalveolar lavage fluid (BAL), and CSF
● Place 5 or 6 drops of sample plus 1 drop of 37% formalin into a cytospin specimen chamber. Follow procedure for operation of
centrifuge from manufacturer.
● As an alternative when the specimen is viscous or cloudy or the quantity is not sufficient for concentration, use a Pasteur pipette to
transfer 1 or 2 drops of the specimen directly to the slide, after marking the location with a wax pencil. Allow the drop(s) to form
one large drop. Do not spread the fluid. Optionally, add a second drop of fluid to the same area to increase the concentration of
fluid for examination
b. Urine specimens
● Place 10 µl of well-mixed, uncentrifuged urine on a glass slide marked with a wax pencil. A ring slide may also be used for ease in
locating the specimen.
● Air dry without spreading

c. Specimens received on swabs

● Request a separate swab for adequate smear preparation
● Roll the swab gently across the slide to avoid destruction of cellular elements and disruption of bacterial arrangements
● Alternatively, when only one swab is received, place the swab in a small amount of saline or culture broth, cap tube, and vortex.
Squeeze the swab against the side of the tube, and use swab to prepare smear
d. Specimens not received on swabs: aspirates, exudates, sputa, etc
● Transfer specimen to the clean slide.
● Select purulent or blood-tinged portions of pus or sputum with a sterile applicator stick, pipette, or wire loop.
● Spread the sample over a large area of the slide to form a thin film.
● For extremely thick or purulent specimens, dilute in a drop of saline on the slide for easier smear preparation. Alternately, place the
specimen on one slide, cover it with second slide, press the slides together, and pull them apart. Remove excess material on the
side of slides with a disinfectant-soaked paper towel.
e. Dried material or very small amounts of clinical specimen
● Emulsify specimen in 0.5 ml of sterile broth. Vortex if necessary.
● Use a sterile Pasteur pipette to transfer 1 drop to a slide.
● Use the pipette tip to spread the drop into an even thin film.
f. Biopsy specimens and tissue sections
● Touch preparation, place tissue in sterile petri dish, and mince with sterile scissors or surgical scalpel. With sterile forceps to hold
pieces, touch the sides of one or more of the minced fragments to a sterile glass slide, grouping the touches together for easier
examination.
● Thin-smear preparation, when specimen sample is soft tissue or thick exudate, prepare smear by taking a small portion of tissue
and placing it on sterile slide with a swab or sterile forceps. Place a second sterile slide over sample, and press slides together.
Separate slides by sliding the two away from each other.
Broth cultures
● Prepare one smear per slide to avoid washing off of “dried” liquid from one area to another during staining.
● Use a sterile Pasteur pipette (or a venting needle or syringe adapter for containers with septa, such as blood culture bottles, to
avoid manipulating a needle and syringe) to transfer 1 or 2 drops to the slide.
● Spread drop into an even thin film.
● For charcoal-containing medium, prepare blood culture smear as for hematology blood count, using a second slide at a 45 angle
to spread a thin layer of cells on first slide.
Colonies from solid media
● Place a drop of sterile saline or water on slide. Distilled water may distort cellular morphology of fragile organisms.
● Transfer a small portion of colony with a sterile applicator stick, wire needle, or loop.
● Gently mix to emulsify.
● The resulting smear should be slightly cloudy and homogeneous
● If swirl lines are evident as the smear dries, the inoculum was too heavy, the drop of saline was too small, and/or the smear was
spread over too small an area.
SMEAR FIXATION
● Air dry slides in a biosafety cabinet or covered on a slide warmer at 60C until dry.
● Heat fixation
- Pass air-dried slides two or three times through a flame, or hold the slide against the front of a microincinerator for 5 to 10 s. To
avoid distortion, do not overheat.
- Allow slide to cool before staining.
● Alternatively, fix with methanol to prevent the lysis of RBCs, produce a cleaner background, and prevent washing off of liquid
specimens.
- Place a few drops of methanol on an air-dried slide for 1 min, drain off remaining methanol without rinsing, and allow the slide to
air dry again.
- Do not heat before staining.
STAINING PROCEDURE
Do not apply stains, water, or decolorizer directly to specimen area. Apply drops near the frosted end of the slide, allowing reagent to
flow over the remaining surface.
● Flood the fixed smear with the crystal violet solution. Allow the stain to remain for 30 s.
● Decant crystal violet, and rinse slide gently with running tap water.
● Rinse off excess water with iodine solution, and then flood the slide with fresh iodine solution. Allow iodine to remain for 30 s.
● Rinse off iodine gently with flowing tap water.
● Decolorize by letting the reagent flow over the smear while the slide is held at an angle. Stop when the runoff becomes clear.
Adjust decolorization time to thickness of smear and type of decolorizer used.
● Remove excess decolorizer with gentle flow of tap water.
● Flood the slide with counterstain and allow to remain for at least 30 s (for ≥1 min with fuchsin stains). Use one of the following
counterstains.
- Safranin
- 0.1 to 0.2% basic fuchsin
- Carbol fuchsin or 0.1 to 0.8% basic fuchsin counterstain for detecting faintly staining Gram-negative organisms
● Remove excess counterstain with a gentle flow of tap water.
● Drain slide, and air dry it in an upright position or use a commercial slide drier. Slides may be gently blotted with filter paper to
remove excess water, but care should be taken to avoid wiping the stained material from the slide.
EXAMINE THE DIRECT SMEAR MICROSCOPICALLY
Evaluate the general nature of the smear under low power (defined as 10 objective with 10 eyepiece)
a. Observe for stain crystals.
● If an excess of precipitated stain is observed, decolorize and restain slide/prepare another Gram-stained smear.
● If precipitate continues, use freshly filtered crystal violet or counterstain in a clean container.
b. Determine if smear has been properly decolorized.
● Depending on the source of the specimen, the background should be generally clear or Gram negative.
● If WBCs are present, they should appear completely Gram negative.
● If slide is overdecolorized, completely decolorize and restain slide, or prepare another Gram-stained smear.
c. Determine if thickness of smear is appropriate. For proper interpretation, areas must be no more than one cell thick, with no
overlapping of cells.
d. For smears prepared from clinical specimens, examine several fields (10 for urine and 20 to 40 for other specimens) under low power
for evidence of inflammation.
● Observe distribution of organisms and cells.
● Determine areas representative of inflammation or purulence and areas of apparent contamination with squamous epithelial cells
(SECs). If no purulence is seen, choose areas of apparent necrosis, inflammatory cell debris, and mucus.
Determine the average count of WBCs and epithelial cells
If cells are present, in 20 to 40 representative fields that contain cells.

Observe cell morphology and Gram reaction

In a representative area with a preponderance of inflammation or purulence using the oil immersion lens (defined as 100 objective with
10 eyepiece), examine 20 to 40 fields.
● If rare or no organisms are seen from a normally sterile-site specimen, but the specimen appears purulent, or the specimen looks
suspicious, perform a more extensive review of the slide.
● Certain critical specimens where low numbers of organisms may be seen (e.g., CSF) should be read more extensively, and the
entire slide may need to be read to substantiate a negative result.
Preservation of direct smear slides
● Drain or gently blot excess oil from slide and save slides for further evaluation for a minimum of 1 week to allow a confirmatory
review, especially if culture or other laboratory test results are inconsistent.
● Should a slide need to be restained to repeat Gram stain or prepare a special stain to confirm findings suggested by a Gram stain,
remove the immersion oil with xylene or a xylene substitute and decolorize smear. Then restain slide.
● For slide libraries and teaching collections that will be stored for longer periods, remove immersion oil with xylene and overlay
smear with a sealer to prevent fading.
POSTANALYTICAL CONSIDERATION
INTERPRETATION
LOW POWER OBSERVATION
● WBCs and RBCs suggest an infectious process. note: neutropenic patients may have few WBCs, but they may have necrotic
debris and protein in the background.
● SECs, food debris, etc., suggest an improperly collected specimen
● The presence of certain microorganisms (e.g., branching hyphae) indicates an infectious process.
OIL IMMERSION OBSERVATION
a. Observe microorganisms for characteristic morphology and
presentation.
b. Gram reaction
● Gram positive: deep violet
● Gram negative: pink or red (carbol fuchsin counterstains have a
more intense color)
● Gram variable: both Gram-positive and Gram-negative cells with
the same morphology. Note: This may result from a smear of
uneven thickness, incomplete decolorization, overdecolorization,
presence of older cells, damage of cell walls, or Gram-variable
nature of the particular organism.
● Gram neutral: taking up neither the crystal violet nor the
counterstain. Note: these cells appear colorless against a generally
Gram-negative background and may be intracellular. This reaction
is seen in smears of clinical specimens when fungal elements or
Mycobacterium spp. are present, both of which have poor uptake of
- The presence of a microorganism from a normally sterile site is likely to indicate infection with that organism.
- Physician offices may perform Gram stains on unspun urine. The presence of microorganisms is likely to indicate a bacterial
count of ≥105 CFU/ml.
- The presence of large numbers of a single type of microorganism in a noninvasively collected specimen, especially if associated
with WBCs, is likely to indicate infection.
- Be wary of interpretations made from observing very few organisms (especially in the absence of inflammation or if the
organisms are unevenly distributed), because collection tubes, slides, and media may harbor nonviable bacteria.
REPORTING RESULTS
● If no organisms or cells are detected in a smear of a clinical specimen, report “No organisms seen” or “No cells seen,”
respectively.
● Determine number of cells and bacteria.
● Notify the caregiver or physician of record, depending on local policy, of any clinically significant findings (any bacteria from a
normally sterile site). Document notification, including date and time of notification.
THANKS
Does anyone have any questions?

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