Identifying and Isolating Microbes from the Environment

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Identifying and isolating microbes from the environment
Abstract
Microbes are found in different locations and surface types, and researchers have in the past relied on culturing microbes to isolate and identify the microbes. Increasingly, DNA sequencing has been utilized as a more effective way to identify microbial characteristics in the built environment different samples were collected in the biology lab and the hallway where gram staining and 16S rRNA sequencing was conducted to identify the various microbes on the surfaces. After analysis, Micrococcus luteus strain, uncultured bacterium clone and staphylococcus sp strains were identified, but the other five could not be identified. Sequential analysis was possible through the use of FINCH and BLAST programs, and gel image was also utilized for analysis. Human contact was identified as a strong predictor of microbes form the built environment, while spatial proximity, the location and the time the study was conducted could also explain microbial diversity.
Introduction
Microorganisms are everywhere in the built environment and culturing the microbes is necessary to identify the microbes present in this environment. Even as this approach has been used over time there is a drawback since some microbes cannot be cultured. DNA sequencing is equally cheap and effective since it is possible to identify the microbes when here are no cultures. As human have created the built environment the microhabitats have become more diverse than ever before, and microorganisms colonize these habitats (Kelley & Gilbert 1). When compared to the natural world, the microbial life of various microorganisms is remarkably different as there are more people in moving in and out of the built environment. The source of the microbes may be human, water and environmental elements, while even insects and animals can transport the microbes.
Even as culture based methods are used to identify and isolate microbes, they underestimate the diversity of microbial communities. The pattern of occupancy influence bacterial communities in the built environment (Stephens et al 1). However, in the case of buildings with moisture problems, the fungal communities thrive even when they are from the outdoor environments, while humans also influence their presence (Stephens et al 1). Additionally, the building characteristics like surface materials, design and ventilation strategies affect the diversity of microbial communities (Meadow et al 2).
In past research the culture based studies have shown that microbes are everywhere in the built environment, but we still do not fully understand microbial diversity (Kelley & Gilbert 1). Isolating and identifying the microbes from the build environment is necessary to understand how the building attributes and biodiversity influence human health. Studies on microbes in the built environment have mostly focused on identifying pathogenic strains, bioaerosol concentrations and the presence of culturable strains (Kembel et al 1469-1470). More research to understand the microbial diversity of the built environment is necessary. This study applies the culture the gram stain and 16S ribosomal sequencing approaches to identify and isolate microbes from the built environment.
Methods
Setting and study design
Different swab samples were taken from different chairs in the biology lab and benches in the hallway on the 3rd floor. Subsequently, the microbes were cultured from the swabs where the species richness was identified to determine microbial diversity. All the samples taken were labeled depending on their source location, and this was necessary to provide a detailed description of the type of microbes depending on their location and the built environment.
Gram staining procedure
The samples were placed on culture plates where the isolated colonies then formed after some time. Gram staining was chosen to determine bacterial morphology and distinguish various cells. Since the colonies contained many bacterial cells, observation was made on colony size, shape, edge, surface, color and bacteria shape. Crystal violet was also added to the staining followed by iodine to stabilize and destain the sample with the bacteria appearing purple. The colonies were then Gram stained, observation made whether they were Gram positive of negative after extraction. Safranin was applied for counterstaining, and the Gram positive bacteria were still purple, while the Gram negative bacteria were pink.
Sequence processing
The DNA was isolated from the microbes, where the cells were first separated from the environment followed by opening the cells and isolating the DNA from the macromolecules. The WVU sequencing center then conducted chain termination sequencing where the DNA Concentration (ng/uL) and 260/280 Ratio were recorded. In evaluating the DNA concentration, the 260/280 ratio was determined and the values recorded to indicate the sequencing reaction. Subsequently, the ratios were compared with the 1.8 and 2.0 range identified as being representative of samples, which have low contaminants. The 260/280 Ratio also indicates the purity when analyzing the DNA or RNA quality, and evaluating whether the purity ratios are high or low indicates whether there was DNA/RNA contamination. This is followed by confirmation that 16S rRNA has been successful after amplification, as represented by the occurrence of a band. This is followed by quantification of the library pool concentration represented (ng/uL). Care is exercised to prevent contamination during nucleic acid extraction, Polymerase chain reaction (PCR) amplification and during the sample collection.
Sequencing of 16S rRNA was identified to sequence data for various bacteria species, and the PCR technique was used to amplify the 16S rRNA gene, to allow DNA sequencing rather than cloning. The PCR based method is useful in isolating the DNA or RNA without requiring cultivation of the microbial species. And since the 16s RNA is large and has conserved and variable regions there are interspecific polymorphisms to provide valid measurements. The 16S rRNA is found in all bacteria, and DNA extraction is the first step where the DNA is isolated from the microbes. Depending on the cluster density, the sequencing process allows for various reads, followed by interpretation based on the taxonomic classification.
Sequencing processing was also conducted for the different microbes with emphasis on 16S rRNA sequencing technique. In conducting DNA sequencing, the chain termination method was utilized based on the polymerase chain reaction (PCR). There was denaturation and the use of dideoxyribonucleotide triphosphates (ddNTPs) compared to the conventional PCR. There are modified ddNTPs are incorporate into PCR, and this then terminates the ddNTPs to create DNA fragments, which are then fluorescently labeled. The ‘FINCH’ xiv program was used to assess the quality of results, and the sequence compared with BLAST xv (basic local alignment search tool).
Results
The aim of the study was to determine the microbes from the built environment, which have been isolated from different sites in the biology laboratory and hallway. Table 1 shows sequencing of the microbes collected from the sample chairs and bench, while table 3 is gel image representing the separated DNA molecules based on their sizes. There were three microbes identified after sequencing, the Micrococcus luteus strain, uncultured bacterium clone and staphylococcus sp. Strain, while the other five species could not be identified. The 16S rRNA sequencing was used to identify the dominant bacteria in the built environment.
Sequencing IDSwab Location DNA Concentration (ng/uL)260/280 Ratio 7Achair 1 in biology lab; left side of room 10.61.597BIGNORE (SEQUENCING DID NOT WORK)4.71.517Cchair 2 in biology lab; left side of room 1.61.137Dchair 3 in biology lab; right side of room 9.51.77Echair 4 in biology lab; right side of room 12.31.847Fbench in hallway, 3rd floor 3.71.837Gchair 5 in biology lab; right side of room 4.21.77
Table 1: Sequencing
The DNA sequencing focused on concentration and the 260/280 ratio. For the six samples the e...