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Posted: February 26th, 2023
The human body is naturally inhabited by a wide variety of microbes, collectively referred to as normal flora. To investigate the diversity of these microbes at different sites of the body, swabs were taken from the skin behind the ear and back of the throat and cultivated on blood agar and mannitol salt agar plates. Based on colony morphology and Gram staining, Staphylococcus Aureus and Escherichia Coli were tentatively identified as the most prominent normal flora cultured from the skin and throat respectively.
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To cultivate normal flora present on human skin and in the throat using differential selective media.
To identify the specific bacteria grown from each region by observing the morphology of the colonies on the agar plates and Gram stained slides.
The human body is inhabited by a wide variety of microbes. In a healthy human internal tissue are normally free of microorganisms whereas surface tissues are in constant contact with environmental organisms and become readily colonized by certain microbial species (Toddar 2005). The mixture of organisms regularly found at any anatomical site is referred to as the normal flora or normal biota. Each body surface has its own characteristic resident biota made up of particular microbial species (Ingraham & Ingraham, 2004). The type of bacteria found in a certain location depends on environmental needs such as ideal temperature, pH, physiology and available nutrients. For example, areas such as the armpit, navel or the back of the throat harbour more microorganisms due to the added moisture, higher body temperature and greater concentration of skin surface lipids (Baron 1996).
To aid in the isolation and identification of individual types of bacteria present in our normal flora specialized growth media can be used. Selective media is used to either encourage or inhibit growth depending on the phenotype of the organism. In addition, differential media can help identify between two closely related bacteria that have small phenotypic differences (Ingraham & Ingraham, 2004). Blood agar and mannitol salt agar are examples of commonly used media that are both selective and differential, aiding in the growth promotion, identification and discrimination of common human normal flora. This study aims to investigate and identify the normal flora diversity found on the human body using these standard microbiology techniques.
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Resident bacteria were sampled from two anatomical sites, the skin behind the ear and the back of the throat. Blood agar and mannitol salt agar plates were used; incubation time was 24 hours at temperature of 37°C. Gram staining tests and haemolysis were applied to detect colonies and identify them.
A number of different colonies were observed on both agar plates following isolation of normal flora from the skin and throat. Table 1 outlines the colony description, blood agar haemolysis and subsequent Gram stain from both anatomical sites sampled. On both plates Cocci bacteria were identified; Gram positive were present at both BA plates, and Gram negative bacteria were only identified at the back of the throat. Types of haemolysis were also different: beta type for sample from the skin, and gamma type for throat sample.
Table 2 presents the findings of normal flora colonies grown on mannitol salt agar. The differences between MSA colonies were more significant than between BA colonies: samples from the back of the throat were Gram negative, and samples from the skin behind the ear were Gram negative. Based on these observations and knowledge of the most abundant normal flora at each site, a preliminary identification of the bacteria isolated was made. The bacteria in the throat is most likely Escherichia Coli and Staphylococcus Aureus bacteria is most likely to be identified at the skin.
Table 1: Colony morphology and Gram stain of resident microbes from the skin and throat, isolated on blood agar.
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Throat
Filamentous flat shaped
Gamma
Cocci
Enterococcus aureus
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Escherichia coli
Gram negative and Gram positive
Skin
Circular convex shaped
Beta
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Cocci
Staphylococcus aureus (25% common)
Streptococcus pyogenes (5% rare)
Gram positive
Table 2: Colony morphology and Gram stain of resident microbes from the skin and throat, isolated on mannitol salt agar.
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Throat
Punctiform flat shaped
No color
Cocci
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Neisseria sp.
Neisseria meningitides
Escherichia coli
Proteus sp.
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Pseudomonas
Aeruginosa
Haemophilus
Influenza
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Spirochetes
Gram negative
Skin
Punctiform flat shaped
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Small pink or red colony
Cocci
Staphylococcus
Epidermidis
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Staphylococcus aureus
Streptococcus pyogenes
Corynebacteria (Bacilli)
Gram positive
To investigate the diversity of normal flora, areas from the skin and throat were sampled and the resident bacteria isolated on blood agar and mannitol salt agar plates, prior to Gram staining.
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BA plates are differential: MSA plates are selective and differential. Cultures grew on each half of the plates. The results obtained at BA and MSA plates are different; this may result from several factors: sampling variations, growth variations and approximateness of estimates produced by Gram staining. For throat swabs, results were negative and positive at BA plate, and only negative at MSA plate; for skin swabs, Gram results were positive at both plates. At both halves of the plates major colonies could be identified.
Generally, it was expected to testify greater variety of bacteria at the throat swab compared to skin swab basing on the difference of environments (humidity, higher temperature, exposure to different microorganisms). During the experiment, a slightly greater diversity was indeed observed. Escherichia Coli was determined as major colony at throat sample because BA plate demonstrated gamma haemolysis and the throat swab shown Gram-negative results both times (and Gram-positive results only at BA plate). Different shapes of colonies also correspond to this identification as E. Coli does not have a particular cell arrangement. Staphylococcus aureus were determined as type of colony for skin swab since of its colony type, beta haemolysis reaction and Gram-positive stain, 25% common. Also, Staphylococcus aureus is common for the normal flora of humans found on nasal passages, skin and mucous membranes (Bauman 2008), In order to make more detailed analysis, it is possible to perform catalase test.
To make a conclusion, the results of the experiment demonstrate the diversity and preliminary identification of common normal flora found resident on the skin and throat.
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