The skin is constantly in contact with environmental microorganism. Therefore, it is colonized by certain microbial species. The indigenous microorganisms present on the skin surface are called skin microflora. Most of the skin microflora is from bacteria. The microorganisms that constitute the skin microflora of the human body are usually harmless. However, some of them are potential pathogens or opportunists. These microorganisms may cause disease under certain circumstances including environment condition, growth factors. The microorganisms that are native to skin are expected to be Staphylococcus viridans, Staphylococcus epidermidis, Bacillus spp., Corynebacterium spp., Micrococcus luteus, yeasts and Streptococcus aureus. In addition, Streptococcus viridans, Staphylococcus epidermidis, Corynebacterium spp., and Bacillus spp. are the most commonly isolated microorganisms from skin. By identify skin microflora, one can understand the causes and consequences of colonization.
Sterile water in test tube
Tryptic soy agar (TSA) plate
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Crystal violet solution
Acetone-alcohol (50:50) mixture
2% Aqueous safranin O
A sterile swab is moistened with the sterile water. Carefully swab the arm.
Inoculate the TSA plate with the swab by gently dabbing the swab on the plate and gently rolling it back and forth over the area. Label the plate. Discard the used swab in the appropriate container.
Incubate the plate 48 hours at 37 oC.
Note the colonial appearance. These microorganisms might be skin microflora.
Flame the inoculating loop by holding it in the flame of a Bunsen burner until it is red-hot. Allow the loop to cool for about 30 to 45 seconds.
Place one small loop of water on the slide.
Flame the inoculating loop again and obtain a trace amount of colony culture from the TSA plate.
Slowly mix the colony culture with the water on the slide. Spread the culture suspension as evenly as possible over an area of 1-2cm2.
Air-dry the film on the slide. A very thin semi-transparent, faint whitish layer is most desirable.
Grasp the slide by the edges near one end with smear side up.
Pass it through the upper portion of the flame for about two or three times.
The fixed smear is now ready for gram staining.
Apply 15 drops of the crystal violet solution to the heat fixed smear. Allow the dye to remain on the slide for 1 minute. Rinse the slide with water.
Apply 20 drops of iodine solution and let stand for 1 minute. Rinse off water.
Add the alcohol and acetone mixture drop by drop to the smear with slide held tilted.
Continue decolorization until no more purple stain is seen to wash from the smear.
Immediately rinse the slide in running water.
Add 2% aqueous safranin O for about 50 seconds. Rinse the slide with water.
Carefully blot the slide to hasten drying.
Position the slide on the stage to bring the specimen into sharp focus by using low power objective (10X).
Rotate the high-dry objective (40X) into position and readjust the fine focus.
Examine the specimen under the oil-immersion objective (100X). Add immersion oil directly on the area of the slide to be viewed. Adjust the iris diaphragm to produce optimum illumination.
Note the morphology and arrangement.
Appearance on TSA Plate
Large, round, white, glistening colony
Round, Irregular shape
Both single and chains
Wrinkled and dry
Club shaped, barred
Large, round, yellow, glistening colony
Both single and tetrads
Large, round, moist colony
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Marked to Standard
Large, round, golden-yellow, glistening colony
From the table, skin miroflora consists of gram positive bacteria. Gram positive bacteria do not lose their primary purple after gram-staining procedure. Counter-staining turns the decolorized gram-positive bacteria to a deeper violet colour.
There are indigenous microorganisms present on the skin surface. Tryptic soy agar (TSA) plate is used for this study as it supports the growth of many organisms. Therefore, TSA is called general purpose media.
There are two genera of bacteria, Straphylococcus and Streptococcus, are frequently present on skin.
The morphology of the microorganisms can be discerns by using microscope under oil-immersion objective. Most of the skin organisms are similar in morphology. However, there are variations due to differences in genetics and ecology. From the table, it can be observed that most of them are either cocci or rods.
Streptococci cells usually grow in chains. The individual cocci elongate on the axis of chain and start dividing. Therefore, long chains of cocci can be seen in Streptococcus viridans due to cells adhere after repeated divisions.
Both Staphylococcus epidermis and Staphylococcus aureus divide in random planes hence forming irregular grapelike clumps. Staphyococcus aureus is the most pathogenic of the staphylococci. It forms golden-yellow colonies, which is protective against the antimicrobial effects of sunlight. Micrococcus luteus divides in two planes to form square groups of four cells called tetrads.
Both Corynebacterium spp. and Bacillus spp. appear as rods. The shape of the rod varies between species hence it is club shaped and barred for Corynebacterium spp. while single and chains for Bacillus spp. Rods usually are single. However, Bacillus spp. remains together after division to form chains.
Yeast form budding cells as expected. There is a rise in cell number when yeast reproduces by budding.
There are some improvements can be made. The simple stain can be performed to make the cells more visible. Methylene blue can elucidate the morphology and arrangement of the organisms. Apart from that, phase-contrast microscope can be used to examine the cells. Some of the cells like yeast can be observed distinctly by using phase-contrast microscope. In addition, selective media can be used to grow the microorganisms after identification of each microorganism to have a deep study on them. Bacteria may be selected by incubation with nutrients that they specifically can use.