Abstract
Background
Microorganisms especially bacteria have occupied a top position in the ecological niche in terms of their distribution. They are reported to be present every where and hence they are better regarded as ubiquitous in nature. The growth inducing properties of these special microbes have been a considerable research interest in laboratories since many years. However, it is unknown whether bacteria are perfectly ‘ubiquitous’ and whether available techniques could address the issues related to contamination, maintenance of cultures, and growth of desirous bacteria.
Methods
A total of three bacteria were included in the study and they are Bacillus subtilis, E. coli and Klebsiella pneumonia. Techniques such as petri plate exposure to air, swab / swab rinse method ,aseptic and non aseptic technique, aseptic inoculation of solid agar media by streak plate technique, serial dilution, isolation of pure culture from mixed population and pour plate and sread plate techniques , colonu enumeration were used to determine the growth parameters that might otherwise reflect the ubiquitous nature of bacteria under study.
Results
The aseptic techniques have very well contributed to the growth of bacteria.
The nutrient agar growth media was found to be well suited to the bacteria.
The characteristic features of all bacteria such as size, shape, color, margin and texture were proper to the bacteria. Inoculation of broth, slopes and plates has equally contributed to the growth patterns of repective bacteria. Test tubes with 10-4 and 10-5 have contributed to more number of E.coli colonies on spread plate compared to pour plate. Conclusion: Bacteria like B.subtilis, E.coli, K.pneumoniae have become well adapted to the sterile conditions maintained in the laboratory. This has indicated that given a perfect aseptic and growth deermining conditions, there could be luxuriant growth of desirous bacteria.This might also eliminate irrelevant bacteria that could interfere experimental conditions and likely affect the outcome. Hence, bacteria may be considered ubiquitous uness further studies are warranted.
Introduction
The flora and fauna of the living world are gifted for their distribution. This could range from deep sea to extraterrestrial environment. The visualization of many living creatures was made feasible through the scientific advancements and ever exploring discoverers. However, the miniature sized creatures are more likely to pose a problem as they are invisible to the naked eye. These are nothing but Microorganisms.
They were considered to be present every where and due to that nature they were referred to as “Ubiquitous”. For example, a thermophilic bacterium known, Thermus acquaticus lives in hot springs (Dubey and Maheswari, 2000). Similarly, a study has described that E. coli counts were more in stream sediments, followed by bank sediments, sediments along spring margins, stream water, and isolated pools, in forest soils (Muruleedhara et al., 2003).
The scientific study of Microorganisms reveals many facts. They are strongly believed to play vital role in contributing to a plethora of infections which could lead to even fatal conditions. The common examples include diphtheria, tretanus, pneumonia etc. They could also cause diseases in animals and plants. On the other hand, the microorganisms also pose a problem to a working environment more probably a laboratory. They could interfere with the outcome of work, maintenance of important lab ware, chemicals and reagents etc. This could be due to their remarkable potential of “Contamination’’ which is strongly connected to their ubiquitous distribution.
They are in the air that circulates around us, food we consume and water we drink, habituate on the surfaces of objects including the skin of our fingers. Due to this, their distribution may be always considered to be complicated and ever ending problem especially for a hygienic and aseptic environment. This alarming situation has the drawn the attention of many investigators to study the distribution of a spectrum of microorganisms. Various concepts have been devised and modulated to isolate and study them from the areas where they are confined. There is a need to know about these strategies.
Therefore, the present description is concerned with highlighting the techniques that are intended to study three types of bacteria Bacillus subtilis, E-coli, and Klebsiella pneumonia obtained from their natural habitat. These include aseptic vs non septic technique, aseptic inoculation of broth culture, aseptic inoculation of solid agar media and streak plate technique, isolation of pure culture from mixed population, preparation of dilution series, spread plate technique and pour plate technique.
Discussion
Firstly, the ubiquity of microorganisms was studied by the swab method. This method is based on the use of a sterile pre-moistened swab to isolate microorganisms from the surfaces. The swab was moistened as it may help in easier picking of good number of bacteria. Similarly, in the swab rinse method the tip of the cotton swab is moistened by placing it in a known volume of sterile buffer/ saline. This saline is subsequently used for determination of bacteria. Here, it is anticipated that the saline solution contains bacteria.
Their growth will be checked when the solution is spread or poured over the sterile Petri plate containing any suitable bacteria supporting growth medium (Nutrient agar). This swabbing technique method yielded 2 colonies.
The first colony has 2mm size, wrinkled shape, and opaque color, irregular margin, elevated with round texture. These features indicate that the plate is harboring colonies typical of Bacillus subtilis. The second colony has 0.5 mm size round shape, yellow color, wavy margin, flat elevation with smooth texture. These features indicate that the plate is harboring colonies typical of E.coli. The plates with nutrient agar medium have well facilitated the growth of these bacteria. This could indicate the efficacy of sterile conditions maintained in the laboratory there by reflecting the aseptic condition.
Secondly, the technique of aseptic vs non aseptic technique involved comparison between a sterile inoculating loop and a non sterilized loop. Here, the inoculating loop was sterilized by inserting it into the hottest part of the flame. This process not only enables perfect sterilization of the loop but also ensures the growth of the desired bacteria. In other words, this method was done to check whether sterilization favors the growth of the bacteria under study.
Similarly, the inoculating loop was allowed to touch the fingers and inserted into a tube containing nutrient agar. This is done to check whether contamination prevents or promotes growth of the bacteria.
As expected, the tube ‘A’ inoculated with non-sterile loop has a turbid color indicating the absence of bacteria. The tube ‘B’ inoculated with sterile loop has a clear yellow color indicating the absence of Bacteria. This result has indicated that aseptic technique is essential for a contamination free environment in the medium containing plates on which bacterial growth was observed. Likewise, the results also indicated the connection between contamination, induced by a non- ascetic method, and absence of bacterial growth on the medium. Next, the aseptic method of inoculation was applied to test tubes harboring various broth cultures. These include controls and that of B.subtilis, E.coli, and K.pneumoniae.
This resulted in absence of any bacterial growth in two control tubes. In the third tube, fine particles were observed in semi cloudy form at the bottom of the tube. This has indicated the growth characteristic of B.subtilis. In the second tube, there were few tiny particles in a little cloudy appearance at the bottom of the tube. This has indicated the growth of E.coli bacteria.
Similarly, tube 5 has cloudy appearance indicating the presence of K.pneumoniae. Therefore these results provide a scientific evidence for the aseptic condition. In another method, agar slopes were prepared and inoculated with sterile loop containing all the three types of bacteria. This is done to observe for the growth patterns. It was revealed that Bacillus subtilis was more luxurious in its growth at the bottom of the tube in the nutrient broth. In the case of slopes, the growth was in zig-zag manner i.e it is more on bottom and less on top. These bacteria ranged from 0.-7 mm in size when observed under electron microscope. Further, that they are circular, brown, wrinkled, convex and smooth in their characteristic features. Similarly, the broth, slopes and plates enriched with nutrient agar also favored the growth of E.coli with patterns that resemble B.subtilis.
However, their size varied from 1-5 mm in diameter and they are all circular, beige colored with entire margin, flat elevation and round texture. The growth for Klebsiella pneumonia also resembled the two other plates i.e high at the bottom and low on the top surface. Their size varied from 1-5 mm in diameter and they are all circular in shape, pinkish in color with lobate margin, hilly elevation and round texture.
These growth patterns may clearly indicate that inoculation could be more on the bottom and minimum on the top. Next, streak plate method is performed by using an inoculation needle which is bend t form loop in order to transfer the microbes from cuture broth.Here, the sterile loop is inserted into a sterile petri plate containing nutrient agar and streaked in zig-zag manner by removing the loop backwards and forwards firmly. Due to this, the cells are deposited evenly and may later develop into independent colonies.
It was reported that microbial growth is determined by many factors (Dubey and Maheswari, 2000).The water availability could be considered as one of the factors influencing the growth of microorganisms (Dubey and Maheswari, 2000). More probably water content of the environment and water soluble salts, and sugars influence the growth(Dubey and Maheswari, 2000). It is reasonable to mention that the ubiquitous nature of microorganisms may be often influenced by the water. For example, some microorganisms show better growth characteristics in water of high salt concentration. These include halophiles such as Pseudomonas spp and Vibrio spp(Dubey and Maheswari, 2000).
Likewise, other microorganisms are in need of high sugar percentage and are called osmophiles such as Saccharomyces, Bacilli and pencillium spp(Dubey and Maheswari, 2000). In the present case, the medium that supported growth of E.coli, B.subtilis and K.pneumoniae could be having enough growth determining parameters. Further, in order to obtain or isolate pure culture from a mixed population of cultures the strategies used may be exposure to air and streaking technique which were already discussed. This has indicated that the growth media (nutrient agar ) used for this process is more likely to favor B.subtilis, E.coli and K.pneumoniae.
This may indicate a kind of selection pressure that may eliminate the growth of irrelevant bacteria. Hence, it is reasonable t mention that no other bacteria were found to be seen after incubation. However, there is also a need of performing a serial dilution technique to perfectly isolate colonies for cell enumeration or counting. This involves arranging a series of glass test tubes continuing distilled water (100ml) and labeling them as 1,2,3….etc. in order to achieve dilution as 1/10,1/100,1/1000,1/10000,1/100000 etc.
Here, a 10 ml of the culture is transferred in to the tube labeled 1 and subsequently 10 ml of the mixture in the tube 1 is transferred to the tube 2. This procedure is continued till the 4th tube. This strategy of dilution is to study and count the presence of bacteria obtained through dilutions in a minute concentration from original pure culture.
The subsequent step is to pour the test tubes containing the dilutions on the corresponding sterile petri plates containing the nutrient agar medium. The colonies that were obtained from the dilutions of tube 4 and 5 on spread plate and pour plate techniques were enumerated. Tube 4 has yielded approximately 200 and 45 colonies on the spread plate and pour plate, respectively. The dilution of Tube 5 resulted in approximately 65 and 60 colonies on spread plate and pour plate, respectively.
This could be due to the growth kinetics that may be more favoring the spread plate compared to pour plate. The sterile glass spreader that was used may be efficient in transferring good number of bacteria and enabling them adhere to the nutrient agar substratum. Pour plate method may ensure uniform suspension but low growth of bacteria may be due to limited suspension and availability of nutrient agar media that is more contributing for the growth. However, this needs further investigation.
Conclusion
The presence of bacteria in the environment is creating an ever increasing problem of contamination. The term “ubiquitous/ ubiquity” may be coined due to the cosmopolitan distribution of microbes. A spectrum of techniques have invaded the laboratory to study the aseptic and septic conditions in order to determine the possible routes of contamination. Petri plates, inoculating loops, nutrient agar medium play essential role in the maintenance of cultures. However, it is the sterilization procedure that better determines the outcome of technique. Hence, its role could not be underestimated in the microbiology. The isolation of pure culture from mixed population has made clear that not all bacteria are subject to luxuriant growth in a given growth inducing condition. This has further indicated that not all bacteria are ubiquitous.
Serial dilution technique has good implications to rule out the enumeration procedure worth fitting for any bacterium which is present at a high concentration at the highest dilution further indicating the population density at a given environment. Therefore, the bacteria, Bacillus subtilis, E.coli and K.pneumoniae have shown remarkable growth characteristic features by adopting to a typical laboratory environment where the conditions were pre studied and informative.
References
Dubey RC, Maheswari DK. 2000. A text book of Microbiology. (1 ed).
Muruleedhara Byappanahalli, Melanie Fowler, Dawn Shively, and Richard Whitman. “Ubiquity and Persistence of Escherichia coli in a Midwestern Coastal Stream.” Appl Environ Microbiol. 69.8 (2003): 4549–4555.
Ubiquity of microorganisms. 2009. Web.