Skillab GUS

Specimen Collection Fakultas Kedokteran Universitas Pasundan

2021

GRAM STAINING OF GENITAL DISCHARGE SPECIMEN

I. GENERAL OBJECTIVE

After finishing skill practice of this session, the student will be able to perform Gram staining

procedure and microscopic examination from the clinical specimen

II. SPECIFIC OBJECTIVE

At the end of skill practice, student could interpret the result of Gram staining from genital

discharge

III. METHODS

▪ Presentation

▪ Demonstration

▪ Coaching

▪ Self practice

IV. LIST OF EQUIPMENT AND REAGENTS

EQUIPMENT

o Bunsen burner

o Object glass/ glass slides

o Smear preparation from genital discharge

o Genital discharge specimen on sterile cotton swab

o Filter paper

o Slide holder/ box or rack

o Microscope

REAGENTS

• Crystal Violet (the Primary Stain)

• Iodine Solution (the Mordant)

• Decolorizer (96% ethanol)

• Safranin (the Counterstain)

• Water (preferably in a squirt bottle)

• Alcohol 70%

• Oil emersion
Skillab GUS
Specimen Collection Fakultas Kedokteran Universitas Pasundan
2021

WHY SHOULD PHYSICIANS EXAMINE GRAM-STAINED SMEARS?

Statistically about one third of all patients admitted to general hospitals have, or develop,

infections, a large proportion of which are caused by bacteria and fungi. In most cases, the

organisms cannot be identified by clinical presentation alone.

Gram stain of collected specimens may help:

1. Make a presumptive etiologic diagnosis and early clinical decisions: Immediate examination
of a Gram-stained smear of material from the infection site can often provide important data
on which to base early clinical decisions, prior to the availability of culture results. The Gram-
stained smear may allow a presumptive etiologic diagnosis to be made within minutes,
whereas culture results usually are not available for one to two days. Early diagnostic
information obtained from Gram-stained smears often allows the physician to prescribe
narrow-spectrum antimicrobial therapy, thereby reducing the risk of toxicity, superinfection,
and the expense of broad-spectrum "poly-pharmacy."

2. Suggest a need for non-routine laboratory procedure: The Gram-stained smear may indicate
a need for laboratory procedures not routinely employed, such as anaerobic and fungal
cultures or special staining techniques, without which the organism might be missed.

3. Help make accurate interpretation of culture results: The Gram-stained smear may provide
clues that are important in interpreting culture results. In patients who have already received
antibiotics, the direct smear may show organisms that will not grow in culture.

4. Provide a better insight into the nature of the current infection: In most cases, the Gram-
stained smear may reflect what is happening in the patient better than a culture. In mixed
infections, due to several types of aerobic and anaerobic bacteria, the smear may indicate the
relative abundance of different bacteria, whereas in culture, the bacteria may grow at
different rates, giving a false quantitative picture. Estimates made regarding the total quantity
of organisms present can sometimes be made from the Gram-stained smear.

SMEAR PREPARATION

Principle Since bacteria are almost colorless, in order to determine the cellular morphology,
stains are necessary. The first important step in staining is to prepare a bacterial smear. This
exercise teaches one technique which is used for the Gram stain. The preparation of a smear is
required for many laboratory procedures, including the Gram stain. A smear can be prepared
from a solid or broth medium, or directly from clinical specimen. Make a thin film of the
material on a clean glass slide, using a sterile loop or swab for viscous specimens.

GRAM-STAINING PROCEDURE

Purpose The Gram stain is the most commonly used differential stain for determining cell

morphology. Differential stains allow for distinguishing certain characteristics of cells, and the

stains commonly use two or more stains. The Gram stain, which divides most clinically
Skillab GUS
Specimen Collection Fakultas Kedokteran Universitas Pasundan
2021

significant bacteria into two main groups, is the first step in bacterial identification.

Gram-staining is a four part procedure which uses certain dyes to make a bacterial cell stand

out against its background. The specimen should be mounted and fixed on a slide before you

proceed to stain it.

Before starting, make sure that all reagents, as well as the squirt-bottle of water, are easily

accessible because you won't have time to go get them during the staining procedure. Also,

make sure you are doing this near a sink because it can get really messy. Wear a lab coat.

No. Step Skor

0 1 2
Smear Preparation from genital discharge
specimen on a swab
1. Clean glass slide by wipes it using cotton
balls soaked with 70% alcohol or burns
it directly over the flame to make the slide
fat free
2. Make a mark as big as thumb nail in the
bottom of the glass slide

3. If the specimen is received on a swab, gently

roles the swab on a cleaned glass
slides to avoid rupturing host cells.

4. Allow the specimens to dry on the slide at

room temperature. Do not heat the
slide to speed drying because this can
distort the cellular morphology or staining
properties of the organism
Skillab GUS
Specimen Collection Fakultas Kedokteran Universitas Pasundan
2021

5. After the specimen has dried, heat-fix the

slide. Gently heat the slide by passing

quickly through the flame, specimen side

up, 3-5 times. It should be warm but not hot
to the touch. The smear is now ready for the
staining procedure.
GRAM-STAINING PROCEDURE
6. Primary stain (crystal violet)
Place your slide on a slide holder or a rack.
Flood (cover completely) the entire smear
with crystal violet. Let the crystal violet
stand for about 60 seconds

7. When the time has elapsed,

wash your slide for 5 seconds with water.
The specimen should appear blue-violet
when observed with the naked eye.

8. Iodine
Flood your slide with the iodine solution. Let
it stand about a minute as well.

9. When time has expired, rinse the slide with

water for 5 seconds and immediately
proceed to step three. At this point, the
specimen should still be blue-violet.

10. Decolorizer (ethanol)

Dip the slide in the 96% ethanol. Step
Skillab GUS
Specimen Collection Fakultas Kedokteran Universitas Pasundan
2021

3 is somewhat subjective because using too

much decolorizer could result in a false
Gram (-) result. Likewise, not using enough
decolorizer may yield a false Gram (+)
results. To be safe, add the ethanol
dropwise until the blue-violet color is no
longer emitted from your specimen.

11. Rinse with the water for 5 seconds.

12. Counterstain (safranin)

The final step involves applying the
counterstain, safranin. Flood the slide with
the dye
as you did in steps 1 and 2. Let this stand for
about a minute to allow the bacteria to
incorporate the safranin. Gram positive cells
will incorporate little or no counterstain and
will remain blue-violet in appearance. Gram
negative bacteria, however, take on a pink
color and are easily distinguishable from the
Gram positives.

13. Rinse with water for 5 seconds to remove

any excess of dye.

14. Blot the slide gently with bibulous

paper or allow it to air dry before viewing it
under the microscope. DO NOT RUB THE
SMEAR!
15. EXAMINE THE FINISHED SLIDE UNDER A
MICROSCOPE.
Observation under light microscope using,
10 x ocular, 100x objective lenses, put 1
drop of immersion oil on the smear
16. Interprete the result
Skillab GUS
Specimen Collection Fakultas Kedokteran Universitas Pasundan
2021

Filamentous and pleomorphic forms may be observed among the Gram (-) rod species. Gram
reaction of the organism may also change after antimicrobial therapy, Gram (+) bacterial may
become gram variable.

Look at areas that are one cell thick only; observation of thick areas will give variable and often
incorrect results. White blood cells and macrophages should stain Gram-negative, whereas
squamous epithelial cells are Gram-positive.

GRAM-POSITIVE BACTERIA

GRAM-POSITIVE COCCI

Clusters: usually characteristic of Staphylococcus spp., such as S. aureus

Chain: usually characteristic of Streptococcus spp., such as S.

pneumoniae, B group streptococci

Tetrad: usually characteristic of Micrococcus spp.

Skillab GUS
Specimen Collection Fakultas Kedokteran Universitas Pasundan
2021
Skillab GUS
Specimen Collection Fakultas Kedokteran Universitas Pasundan
2021

Gram positive bacilli

Thick: usually characteristic of Clostridium spp., such as C. perfringens, C. septicum, C.

tetanomorphum

Thin: usually characteristic of Listeria spp.

Branched: usually characteristic of Actinomycetes and Nocardia , such as A. israelii Note:

Mycobacteria are not branched, and often stain poorly with Gram stain. Some mycobacteria do
appear as Gram-positive rods, not unlike diptheroids, sometimes with Gram-positive beading.

Gram negative bacteria

Gram-negative cocci

Diplococci: usually characteristic of Neiseria spp., such as N. gonorrhoeae

In addition, Moraxella spp. and Acinetobacter spp.are often diplococcal in morphology.

Acinetobacter can be pleomorphic, and sometimes appear as Gram-positive cocci.

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Skillab GUS
Specimen Collection Fakultas Kedokteran Universitas Pasundan
2021

Coccobacilli: usually characteristic of Acinetobacter spp., which can be either Gram-positive

or Gram-negative, and is often Gram-variable.

Gram-negative bacilli

Thin rods: usually characteristic of enterobacteriaceae, such as E. coli

Coccobacilli: usually characteristic of Haemophilus spp., such as H. influenzae

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Skillab GUS
Specimen Collection Fakultas Kedokteran Universitas Pasundan
2021

Curved: usually characteristic of Vibrio spp.; Campylobacter spp., such as V. cholerae

C. jejuni

Thin needle shape: usually characteristic of Fusobacterium spp.

Gram positive cocci Gram negative rods

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Skillab GUS
Specimen Collection Fakultas Kedokteran Universitas Pasundan
2021

Observe and try to find: intra cellular gram negative diplococci

and/ or outside polymorphonuclear cells (extracellular)
References:

Hashimoto T, & Birch WX., Department of Microbiology & Immunology Loyola University

Medical Center, Loyola University Chicago. Last Reviewed: February 19, 2003

Pierce & Leboffe, Exercises for the Microbiology Laboratory, and A Photographic Atlas for the

Microbiology Laboratory, 2nd Ed.

Forbes, BA, Sahm, DF, Weissfeld, AS. Bailey & Scott’s Diagnostic Microbiology, 12th Ed., Mosby

Inc, 2007

Murray, Patrick R.; Baron, Ellen Jo; Pfaller, Michael A.; Tenover, Fred C.; Yolken, Robert H.:

Manual of Clinical Microbiology, 9th Edition, ASM Press 2007

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