1.

PROCEDURES
A. Collecting Bacteria Samples
i.

The lid of the prepared petri dishes was removed from one of the plates.
Prepared petri dishes should be refrigerated until used and always stored
upside down. This keeps condensation which forms in the lid from dropping
onto and disrupting the bacteria growing surface.

ii.

Collect bacteria from each location using one swab for each new spot. The
bacteria can be sampled from a doorknob, the surface of the window and on
the floor.

iii.

Each swab is then streaked across the entire surface of the medium in a petri
plate without tearing into it.

iv.

Cover on each dishes were replaced, tape closed, labelled and incubated in an
inverted position.

B. Preparation of Cell Smear

i.

Preparation of the glass microscope slide :

Cleaned slide are essential for the preparation of microbial smears. Grease or oil from
the fingers on slides were removed by washing the slide with soap and water or
scouring powders, followed by a water rinse and rinsed of 95% alcohol. After
cleaning, the slides was dried and placed on laboratory towels until ready for use.

ii.

Preparation of smear :
Thick and dense smears were avoided. A thick or dense smear occurs when too much
of the culture is used in its preparation which concentrates a large number of cells on
the slide. This type of preparation diminishes the amount of light that can pass
through and makes it difficult to visualize the morphology of single cell.

iii.

Heat fixation :
Unless fixed on the glass slide, the cell smear will wash away during staining
procedure. This is avoided by heat fixation, during which the bacterial protein are
coagulated and fixed to the glass surface. Heat fixations performed by rapid passage
of the air dried smear two or three time over the flame of the Bunsen burner.

C. Simple Staining
Bacterial stain with single reagent produces a distinctive background. The purpose of
this stain is to elucidate the morphology and arrangement of bacteria cell.
i.

Separate smear of the sample microbial given was prepared following the procedure
in B (ii). All smears were heat fixed prior to staining.

ii.

A slide was placed on the staining tray and the smear was flood with one of the
indicated stains, using appropriate exposure time for each: carbol fuchsin 15 30
seconds; crystal violet, 20 60 seconds, methylene blue, 1-2 minutes.

iii.

The smear gently washed with tap water to remove excess stain. The slide was held
parallel to the stream of water in order to reduce the loss of organisms from the
preparation.

iv.

The sample bold dried using bibulous paper but not to wipe the slide.

v.

The stained slides were examined using oil immersion microscope.