Ateneo de Zamboanga University

COLLEGE OF NURSING

NURSING SKILLS OUTPUT Report No. 1

EAR DROPS INSTILLATION AND EAR IRRIGATION

I. DESCRIPTION
Gram staining is a quick procedure used to look for the presence of bacteria in
tissue samples and to characterise bacteria as Gram-positive or Gram-negative,
based on the chemical and physical properties of their cell walls. The Gram stain
should almost always be done as the first step in diagnosis of a bacteria infection.
The Gram stain is named after the Danish scientist Hans Christian Gram (1853
1938), who developed the technique in 1882 and published it in 1884 as a technique
to discriminate between two types of bacteria with similar clinical symptoms:
Streptococcus pneumoniae (also known as the pneumococcus) and Klebsiella
pneumoniae bacteria.

II. MATERIALS/EQUIPMENT NEEDED:







III. PROCEDURE:
1. Get a tissue sample for the Gram stain. For example, if a pneumonia is
suspected, get a sputum sample. If an urinary tract infection is suspected, get a
urine sample. If an intestinal infection is suspected, get a stool sample. If a brain
infection is suspected, get a cerebrospinal fluid (CSF) sample (centrifuge the fluid
and obtain the sediment for the Gram stain). If a skin infection is suspected, get
a skin biopsy sample. If an ear infection is suspected, get a middle ear fluid
sample (to increase yield, or the likelihood of finding the bacteria, centrifuge the
middle ear fluid sample to allow it to settle into three layers: supernatant (top),
buffy coat (middle), and red cells (bottom); get the buffy coat layer for the Gram
stain). Essentially any human tissue can be obtained for the Gram stain.
2. Add 1-2 drops of the tissue sample onto a glass slide. Spread it evenly on the
slide to form a thin smear, which can be done by sliding the edge of another
glass slide across the glass slide containing the tissue sample. Allow it to air dry.
3. Heat fix the smear, by quickly passing it two to three times through a flame, or
heat it on top of an electric slide warmer. Do not overheat, to avoid distortion.
Alternatively, the smear may be fixed by methanol instead, by adding 1-2 drops
of methanol onto the dried smear, draining off excess methanol, and allowing it
to air dry. Methanol has the advantage that it does not lyse red cells and it
minimises damage to host cells, giving a cleaner background.
Bunsen burner
alcohol-cleaned microscope slide
water
Crystal violet, Gram's iodine solution, acetone/ethanol (50:50 v:v),
0.1% basic fuchsin solution
4. Flood the smear with crystal violet. Wait thirty seconds. Crystal violet (CV)
dissociates in aqueous solutions into CV+ and chloride (Cl) ions. These ions
penetrate through the cell wall and cell membrane of both gram-positive and
gram-negative cells. The CV+ ion interacts with negatively charged components
of bacterial cells to stain the cells purple.
5. Gently rinse off the crystal violet with tap water. Do not rinse excessively,
which might remove the stain from Gram positive bacteria.
6. Flood the smear with iodine. Leave for at least three seconds. Iodine, in the form
of negatively charged ions, interacts with CV+ to form large complexes of crystal
violet and iodine (CVI complexes) within the inner and outer layers of the cell.
Iodine acts as a trapping agent to retain the purple crystal violet colour in the
cell.
7. Gently rinse off the iodine with tap water.
8. Decolorize by adding alcohol or acetone to the smear while holding the slide at
an angle to allow the decoloriser to drain. Stop when runoff becomes clear,
within seconds. The decolorisation step is critical and must be timed correctly. If
left on too long, the decolorising agent will remove the crystal violet stain from
both gram-positive and negative cells.
9. Gently rinse off excess decoloriser with tap water.
10. Flood the smear with safranin counterstain. Wait thirty seconds. Counterstain is
applied last to stain the decolorised gram-negative bacteria a pink or red
shadeBasic fuchsin, which stains anaerobic bacteria more intensely, may be
substituted for safranin, but it is less commonly used.
11. Gently rinse off excess safranin with tap water.
12. Drain slide and allow it to air dry. The Gram stain is done.
13. Examine the slide under the light microscope. Gram-positive bacteria appear
purple as stained by crystal violet, which is trapped within their thick cell walls.
Gram-negative bacteria appear pink as stained by the safranin counter-stain, as
their thin cell walls allow the crystals violet to wash out during decolorisation.
Bacteria are further classified by their shape under the microscope, most
commonly as cocci (spherical) or rods (cylindrical).

IV. DIAGRAM/ILLUSTRATION:







V. NURSING RESPONSIBILITIES:
Before Procedure:
During Procedure:
After Procedure:

REFERENCE:
http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/Gram2.htm
http://www.wikihow.com/Gram-Stain





DATE CLINICAL INSTRUCTOR


MICHELLE ERIKA F. MEJIA
BSN III- B