AIM: Study of normal microflora of skin by swab method

PRINCIPLE: The normal floras are regularly found in specific areas of the body. This specifity is far from
arbitrary and depends on environmental factors such as pH, oxygen concentration, amount of
moisture present, and types of secretions associated with each anatomical site. Staphylococci
(predominantly S.epidermidis), Streptococci (α-hemolytic, nonhemolytic, and enterococci),
diphtheroid bacilli, yeasts and fungi. Skin represents sources of mixed microbial populations
from which streak plate inoculations are done for their seperations, study morphology,
biochemistry & microscopic identification to identify individual genera of this mixed flora.

The procedure used to identify the native flora of skin involves the following steps:
a) Blood Agar Plate inoculated to determine the presence of hemolytic microorganisms,
specifically the Staphylococci and Streptococci: differentiation between two genera can be
made based on their colonial and microscopic appearances. The Streptococci typically forms
pinpointed colonies on blood agar whereas Staphylococci form larger pinhead colonies that
might show a golden coloration.
b) Mannitol Agar Plate inoculated for the isolation of the staphylococci: the generally avirulent
Staphylococci species can be differentiated from the pathogenic S.aureus because the latter is
able to ferment mannitol causing yellow coloration of this medium surrounding the growth.
c) Sabouraud Agar Plate inoculated to detect yeasts and molds: Yeast cells develop pigmented
or nonpigmented colonies that are elevated, moist and glistening. Mold colonies will appear as
fuzzy, powdery growths arising from a mycelia mat in the agar medium.

MATERIALS: Blood agar plates, mannitol salt agar plates, Sabouraud agar plates, sterile saline tubes 5ml,
lactophenol cotton blue for fungal staining, crystal violet, Gram’s iodine, safranin for gram
staining, Cotton swabs, microscope, glass slides, Bunsen burner, disposable gloves, glassware
markers.

PROCEDURE: 1) A sterile cotton swab moistened in sterile saline will be used to obtain specimen from
the skin by rubbing the swab vigorously against the palm of hands.
2) Swab will be used to inoculate a tube of sterile saline and the solution mixed.
3) One plate of blood agar, mannitol salt agar, and Sabouraud agar to be inoculated from the
solution prepared in sterile saline by means of a four-way streak inoculation.
4) Sabouraud agar plate have to be incubated for 48 hours at 25°C and the remaining plates for
48 hours at 37°C.

OBSERVATION & DISCUSSION: Blood agar plate will be examined for zones of hemolysis.
Sabouraud Agar plate will be examined for the appearance of moldlike growth.
Mannitol Salt agar plate will be examined for colonies with yellow color medium surrounding
the colonies. This is indicative of S.aureus
Gram stained smear will be prepared from the blood agar culture well defined isolated colonies
and morphologies observed.
Lactophenol-cotton-blue-stained smears of organisms obtained from discrete colonies on
Sabouraud agar culture will be prepared and morphologies observed.
The images shown are for reference of morphologic characteristics on different mediums.
Observations are to be compared with the given reference images.