CONTENTS

1. STANDARD PRECAUTIONS IN HEALTH CARE

2. SPECIMEN COLLECTION

3. CULTURE MEDIA

4. KOH STAINING

5. GRAM STAIN

6. WET MOUNT

7. ANTIBIOTIC SUSCEPTIBILITY TESTING

8. BIOMEDICAL WASTE MANAGEMENT

KOH STAINING
Introduction
 Potassium Hydroxide (KOH) Solutions are used in the preparation of temporary wet
mounts for the direct examination of specimens for fungi.
 Specimens that are mucoid or contain keratin, such as skin, nails, or hair, contain
elements that may obscure or mask fungal elements. KOH has a clearing effect on most
clinical samples by dissolving proteinaceous cellular material and background keratin
allowing for increased visibility of fungal elements. The specimen may be any type of
clinical material, although fluids such as CSF generally do not need to be treated. Also,
the KOH Solutions can be used in conjunction with some fungal stains, such as
Lactophenol Blue Stain (Cat# SL18) or Calcofluor White Stain (Cat# SC15), to help
distinguish fungal elements.
Procedure to make 100 ml of KOH 10% w/v solution
1. Weigh 10 g potassium hydroxide (KOH) pellets.
2. Transfer the chemical to a screw-cap bottle.
3. Add 50 ml distilled water, and mix until the chemical is completely dissolved. Add
remaining distilled water and make the volume 100 ml.
4. Label the bottle and mark it as corrosive. Store it at room temperature. The reagent is
stable for up to 2 years.
5. Label the date of preparation.
Recommended Procedure
1. Place the material to be examined onto a clean glass slide.
2. Add a drop of KOH solution to the material and mix. (If desired, a staining agent may be
added at this point)
3. Place a cover slip over the preparation, and remove any excess fluid by gently pressing
the slide, coverslip down, onto paper towels or blotting paper.
4. Allow the preparation to stand at room temperature until the material has cleared. The
wait time varies between five and thirty minutes depending on the sample.
5. If desired, the preparation can be gently heated to accelerate clarification. (Heat only, if
necessary, as overheating may distort or destroy fungal elements)
6. Examine preparation using a bright-field or phase-contrast microscope. Fungal structures
such as hyphae, yeast cells, spherules, and granules may be distinguished. For bright-
 field, examine slides with reduced light (narrow the iris diaphragm). If slides are negative,
examine slides on several consecutive days.
Interpretation of Results
 Experience is required in examining for fungal elements as background artefacts often
give confusing results
 Hyaline fungi may be difficult to see if illumination is improperly adjusted.
 Avoid direct contact -- potassium hydroxide solutions are extremely caustic and can cause
severe burns or irritation on contact.
 KOH slide preparations are not permanent since even fungi will eventually be destroyed
by potassium hydroxide.
 If a noticeable, heavy precipitate forms discard the KOH solution
Storage and Shelf Life
Our Potassium Hydroxide Solutions should be stored at room temperature and protected
from light. Under these conditions, they have a shelf life of 52 weeks from the date of
manufacture.
References
1. Microscopic potassium hydroxide preparation by David Ponka, Faisal Baddar. Canadian
Family Physician. 2014 Jan; 60(1): 57.
2. Lehmann PF. PR Murray, EJ Baron, MA Pfaller, FC Tenover and RH Yolken, eds.
Manual of Clinical Microbiology.

1.
GRAM STAIN

Introduction
The Gram staining is one of the most crucial staining techniques in microbiology. It gets its
name from the Danish bacteriologist Hans Christian Gram who first introduced it in 1882,
mainly to identify organisms causing pneumonia. Often the first test performed, gram
staining involves the use of crystal violet or methylene blue as the primary color. The term
for organisms that retain the primary color and appear purple-brown under a microscope is
Gram-positive organisms. The organisms that do not take up primary stain appear red under a
microscope and are Gram-negative organisms.
Specimen Collection
Various clinical specimens can be used to perform Gram staining. Some of the commonly
used specimens are sputum, blood, cerebrospinal fluid, ascitic fluid, synovial fluid, pleural
fluid, and urine, etc. Swabs from nostrils, throat, rectum, wound, and cervix, etc. can also be
used. The collection of specimens should always be in sterile containers.
Types of equipment needed for Gram staining include:
 Bunsen burner
 Alcohol-cleaned microscope slide
 Slide rack
 Microscope
Reagents needed for Gram staining include:
 Crystal violet (primary stain)
 Gram's iodine solution (the mordant)
 Acetone/ethanol (50:50 v:v) (the decolorizer)
 0.1% basic fuchsin solution (the counterstain)
 Water
Procedure
1. Preparation of a slide smear:
 Inoculation loop is used to transfer a drop of suspended culture to the microscope
slide.
 If a Petri dish or a slant culture tube has the colony, a drop or a few loopful of water is
added to facilitate a minimal amount of colony transfer to the examination slide.
  A minimal amount of culture is required. If culture can be detected visually on an
inoculation loop, it indicates the collection of too much culture.
 Culture is spread with an inoculation loop to an even thin film over a circle of 15mm
in diameter. A typical slide can contain up to 4 small smears if examining more than
one culture.
 The slide can be either air-dried or dried with the help of heat over a gentle flame. The
slide should be moved circularly over the flame to prevent overheating or forming of
ring patterns in the slide. The heat helps the cell adhesion to the glass slide and
prevents the significant loss of culture during rinsing.
2. Gram staining:
 Crystal violet stain is added over the fixed culture.
 After 10 to 60 seconds, the stain is poured off, and the excess stain is rinsed with
water. The goal is to wash off the stain without losing the fixed culture.
 Iodine solution is used to cover the smear for 10 to 60 seconds. This step is known as
"fixing the dye." Iodine solution is poured off, and the slide is rinsed with running
water. Excess water from the surface is shaken off.
 A few drops of decolorizer is added to the slide. Decolorizers are often the mixed
solvent of ethanol and acetone. This step is known as "solvent treatment." The slide is
rinsed with water in 5 seconds. To prevent excess decolorization in the gram-positive
cells, stop adding decolorizer as soon as the solvent is not colored as it flows over the
slide.
 The smear is counterstained with basic fuchsin solution for 40 to 60 seconds. The
fuchsin solution is washed off with water, and excess water is blotted with the
bibulous paper. The slide can also be air-dried after shaking off excess water.

3. Microscopic examination of slide:

 The slide should undergo an examination under a microscope under oil immersion.
 The initial slide examination should use the X40 objective to evaluate the smear
distribution, and then they should be examined using the X100 oil immersion
objective.
 All areas of the slide require an initial examination. Areas that are only one cell thick
should be examined. Thick areas in slides often give variable and incorrect results.
 White blood cells and macrophages stain Gram-negative.
  Squamous epithelial cells stain Gram-positive.
Various modifications of gram staining are used, such as Atkin gram stain, and Burke gram
stain, etc.
Indications
Gram staining is indicated whenever a bacterial infection is suspected for easy and early
diagnosis.
Potential Diagnosis
Gram staining aids in the diagnosis of a disease or a pathologic condition.
Examples of gram-positive organisms are:
 Cocci: Staphylococcus species, and Streptococcus species
 Bacilli: Corynebacterium species, Clostridium species, and Listeria species
Examples of gram-negative organisms are:
 Cocci: Neisseria gonorrheae, Neisseria meningitidis, and Moraxella species
 Bacilli: Escherichia coli, Pseudomonas species, Proteus specie and
Klebsiella species
Examples of gram variable organisms include:
 Actinomyces species

Normal and Critical Findings

A normal finding in a sterile body fluid should be the absence of any pathologic organism in
the smear. The organisms are identified based on color and shape. Gram-positive organisms
are either purple or blue in color, while gram-negative organisms are either pink or red in
color. Bacilli are rod-shaped, while cocci are spherical.

Findings on gram stain that suggest underlying bacterial infections:

 Gram-positive cocci in clusters: Example- Staphylococcus species such as S. aureus.
 Gram-positive cocci in chains: Example- Streptococcus species such as S.
pneumoniae, B group streptococci
 Gram-positive cocci in tetrads: Example- Micrococcus spp.
 Gram-positive bacilli, thick: Example- Clostridium spp., such as C. perfringes, C.
septicum.
 Gram-positive bacilli, thin: Example- Listeria spp.
 Gram-positive bacilli branched: Example- Actinomyces and Nocardia.
 Gram-negative diplococci: Example- Neisseria spp., such as N. meningitidis.
(Please note: Moraxella spp., and Acinetobacter spp., are often diplococcal in morphology.
Acinetobacter can sometimes appear as Gram-positive cocci, and they can be pleomorphic.
Coccobacilli: Usually characteristic of Acinetobacter spp., and they can be gram-positive,
gram-negative, or gram variable.
 Gram-negative bacilli, thin: Example- Enterobacteriaceae, such as E. coli.
 Coccobacilli: Example- Hemophilus spp., such as H. influenzae.
 Curved: Example- Vibrio spp; Campylobacter spp., such as V. cholerae, and C. jejuni.
 Thin needle shape: Example- Fusobacterium spp.
Gram variable organisms: these organisms do not group into either gram-positive or gram-
negative organisms.

Interfering Factors
If the specimen collection is not sterile, multiple organisms can contaminate the specimen.
Similarly, improper specimen collection and prior use of antibiotics can interfere with the
growth of organisms. During the interpretation of the Gram stain, as described by the World
Health Organization in 2003, the following steps should be followed:
1. General nature of the smear requires analysis under low power magnification (10X)
 The background of the slide should generally be gram-negative or clear.
 White blood cells when present should stain gram-negative.
 Thin crystal violet or gentian violent precipitates should not be confused as gram-
positive bacillus bacteria.
 The smear should be one cell thick with no overlapping of cells.
2. Low power magnification should be utilized to note the following:
 Relative numbers of polymorphonuclear neutrophils (PMNs), mononuclear cells and
red blood cells (RBCs).
 Relative numbers of squamous epithelial cells, and normal microbiota bacteria.
 Location, arrangement, and shape of the organisms.
3. Oil immersion examination of multiple fields is necessary to note the following:
 Micro-organisms: If identified, please note numbers and morphology.
 Shapes: coccus, bacillus, coccobacillus, filaments, and yeast-like.
 The appearance of ends: rounded, tapered, concave, clubbed and flattened.
 The appearance of sides: parallel, ovoid, irregular or concave.
 The axis of the organism: straight, curved or spiral.
 Pleomorphism (variation in shape).
  Branching or cellular extensions.

Complications
The interpretation of slides can be difficult if the microscopic smear is thick and clumped.
Decolorization time should have very close monitoring to avoid under-decolorization or over-
decolorization. Thicker smears require longer decolorizing time. Similarly, cultures should
undergo evaluation while they are still fresh. Old cultures tend to lose the peptidoglycan cell
walls, which predisposes gram-positive cells to be gram-negative or gram variable. Gram
stain is not useful for organisms without a cell wall like Mycoplasma species, and for smaller
bacteria like Chlamydia and Rickettsia species.
Gram stain may not falsely reveal organisms in the following scenario:
 Use of antibiotics before collecting a specimen.
 Inappropriate age of culture: too young or too old.
 Fixing the smear before its dry.
 The smear is too thick.
 Low concentration of crystal violet.
 Excessive heat fixation.
 Excessive washing between steps.
 Insufficient exposure to iodine.
 Prolonged decolorization.
 Excessive counterstaining.
 Lack of experience in preparing the slide, and reviewing the slide.
Sometimes results of Gram-stain may not match the final results of cultures and could
potentially lead to inappropriate use of antibiotics.
Clinical Significance
Gram stain is often the initial diagnostic test for the evaluation of infections. The use of Gram
stain facilitates the rapid use of appropriate antibiotics. However, genetic sequences and
molecular techniques are more specific than classic gram stain.
References
1. Tripathi N, Sapra A. Gram Staining. [Updated 2023 Aug 14]. StatPearls [Internet].
Treasure Island (FL): StatPearls Publishing; 2024 Jan.
2. Bartholomew JW, Mittwer T. The gram stain. Bacteriological reviews. 1952
Mar;16(1):1-29.
3. O'Toole GA. Classic spotlight: How the gram stain works. Journal of bacteriology.
2016 Dec 1;198(23):3128-.
4. Beveridge TJ, Davies JA. Cellular responses of Bacillus subtilis and Escherichia coli
to the Gram stain. Journal of bacteriology. 1983 Nov;156(2):846-58.
WET MOUNT

INTRODUCTION
The Wet Mount is a procedure performed in the laboratory to determine whether or not a
micro-organism is motile and to present unstained material for phase contrast [Link]
is commonly used to examine material collected from the vaginal wall of a female patient.
METHODS
(a) Use of plain slides.
A drop of bacterial suspension is placed in the centre of a clean slide and a cover glass
lowered onto it. It is not necessary to do this carefully since the inclusion of a few air bubbles
in the aqueous film will assist both in locating micro-organisms under the microscope and,
more significantly, in stimulating and retaining active motility in some aerobic organisms.
The resulting thin film of bacterial suspension will be suitable for observation but it will tend
to dry quickly and unless the preparation is to be examined and discarded immediately the
preparation should be sealed by placing a few dabs of vaseline at the corners and along the
edges of the cover slip.
The preparation is then examined under the phase contrast or ordinary light microscope.
(b) Use of cavity slides.
A clean cover slip is placed on the bench and a small blob of vaseline placed in each
corner. A small drop of bacterial suspension is carefully deposited in the centre of the
cover slip.
A well slide is then inverted and lowered over the cover slip so that the vaseline forms an
attachment between the cover slip and the plain area of the slide outside the cavity.
The preparation is then rapidly inverted so that the drop hangs down from the cover slip
into the cavity.
The preparation may be examined using phase contrast or conventional illumination.
Fig. shows hanging drop

A B C
D E F

A-Using a toothpick, place a small dab of Vaseline in each corner of a clean cover glass.
B-Aseptically transfer a loopful of a liquid bacterial culture of the cover glass.
C-Do not spread the drop.
D-Well slide upside down.
E-Carefully lower the slide with depression facing down.
F-Vaseline attaching cover glass to well slide.

References
1. Norris JR, Swain H. Chapter II Staining Bacteria. InMethods in Microbiology 1971 Jan 1
(Vol. 5, pp. 105-134). Academic Press.