Cultivation of Viruses. Nutrition, cultivation and isolation of bacteria. Identification of bacterial pathogens. Bacterial staining methods. Related Books Free with a 30 day trial from Scribd. Dry: A Memoir Augusten Burroughs. Related Audiobooks Free with a 30 day trial from Scribd. Empath Up! Preeti Pandey. Chandani Maurya. Pied Piper. Rani Upadhyay. Pushpa Latha. Saranjack Saranjack. Kalyani Nagasuri. Jeyendra Mamillapalli. Muhammad Hassan. Tonyraj Tonyroyy. Peter Shayo , Student at muhas.
Show More. Views Total views. Actions Shares. No notes for slide. Cultivation of bacteria and culture methods 1. Cultivation of Bacteria 2. Culture Media An artificial culture media must provide similar environmental and nutritional conditions that exist in the natural habitat of a bacterium. The pH of the medium must be set accordingly. A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another.
All glassware, media and instruments must be sterilized i. Consistency 2. Nutritional component 3. Functional use 9. Selective media allows for the growth of specific organisms, while differential media is used to distinguish one organism from another.
There are many types of media used in the studies of microbes. Two types of media with similar implying names but very different functions, referred to as selective and differential media, are defined as follows.
Selective media are used for the growth of only selected microorganisms. Media lacking an amino acid such as proline in conjunction with E. Selective growth media are also used in cell culture to ensure the survival or proliferation of cells with certain properties, such as antibiotic resistance or the ability to synthesize a certain metabolite.
Normally, the presence of a specific gene or an allele of a gene confers upon the cell the ability to grow in the selective medium. In such cases, the gene is termed a marker. Selective growth media for eukaryotic cells commonly contain neomycin to select cells that have been successfully transfected with a plasmid carrying the neomycin resistance gene as a marker.
Gancyclovir is an exception to the rule as it is used to specifically kill cells that carry its respective marker, the Herpes simplex virus thymidine kinase HSV TK. Some examples of selective media include:. Non-selective versus selective media. While the plate on the right selectively only allows the bacteria Neisseria gonorrhoeae, to grow white dots.
Differential media or indicator media distinguish one microorganism type from another growing on the same media. This type of media is used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria.
Examples of differential media include:. Microbiologists rely on aseptic technique, dilution, colony streaking and spread plates for day-to-day experiments. Microbiologists have many tools, but four relatively simple techniques are used by microbiologists daily, these are outlined here.
Aseptic technique or sterile technique is used to avoid contamination of sterile media and equipment during cell culture. This technique involves using flame to kill contaminating organisms, and a general mode of operation that minimizes exposure of sterile media and equipment to contaminants. Serial Dilution : Example of Serial dilution of bacteria in five steps.
The diluted bacteria were then spread plated. When working with cultures of living organisms, it is extremely important to maintain the environments in which cells are cultured and manipulated as free of other organisms as possible. This means passing rims and lids through the flame produced by a Bunsen burner in order to kill microorganisms coming in contact with those surfaces.
Sterile technique, in general, is a learned state-of-being, or mantra, where every utilization of any sterile material comes with the caveat of taking every precaution to ensure it remains as free of contaminants as possible for as long as possible. A serial dilution is the step-wise dilution of a substance in solution. Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion.
A ten-fold serial dilution could be 1 M, 0. A culture of microbes can be diluted in the same fashion. For a ten-fold dilution on a 1 mL scale, vials are filled with microliters of water or media, and microliters of the stock microbial solution are serially transferred, with thorough mixing after every dilution step. The dilution of microbes is very important to get to microbes diluted enough to count on a spread plate described later.
Streak plate : Four streak plates. Successful streaks lead to individual colonies of microbes. In microbiology, streaking is a technique used to isolate a pure strain from a single species of microorganism, often bacteria. Samples can then be taken from the resulting colonies and a microbiological culture can be grown on a new plate so that the organism can be identified, studied, or tested.
The streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop. This is dipped in an inoculum such as a broth or patient specimen containing many species of bacteria. The sample is spread across one quadrant of a petri dish containing a growth medium, usually an agar plate which has been sterilized in an autoclave. Choice of which growth medium is used depends on which microorganism is being cultured, or selected for. Growth media are usually forms of agar, a gelatinous substance derived from seaweed.
Spread plates are simply microbes spread on a media plate. Microbes are in a solution, and can be diluted. They are then transferred to a petri dish with media specific for the growth of the microbe of interest. Anaerobic chambers can also be used within closed compartments, and technicians can manipulate culture media within these chambers.
To encourage carbon dioxide formation, a candle can be burned to use up oxygen and replace it with carbon dioxide. Special microbial media. Certain microorganisms are cultivated in selective media. These media retard the growth of unwanted organisms while encouraging the growth of the organisms desired. For example, mannitol salt agar is selective for staphylococci because most other bacteria cannot grow in its high-salt environment.
Another selective medium is brilliant green agar , a medium that inhibits Gram-positive bacteria while permitting Gram-negative organisms such as Salmonella species to grow. Still other culture media are differential media. These media provide environments in which different bacteria can be distinguished from one another. For instance, violet red bile agar is used to distinguish coliform bacteria such as Escherichia coli from noncoliform organisms.
The coliform bacteria appear as bright pink colonies in this media, while noncoliforms appear a light pink or clear. Certain media are both selective and differential.
For instance, MacConkey agar differentiates lactose-fermenting bacteria from nonlactose-fermenting bacteria while inhibiting the growth of Gram-positive bacteria. Since lactose-fermenting bacteria are often involved in water pollution, they can be distinguished by adding samples of water to MacConkey agar and waiting for growth to appear.
In some cases, it is necessary to formulate an enriched medium. Neither incubation time nor growth medium had an influence on the recovery of rare species. However, we did find differences in time until visible growth on the plate between different phylogenetic classes of the isolates. These results indicate that rare cultivable species are active and not more likely to be dormant than abundant species, as has been suggested as a reason for their rarity.
Moreover, future studies should be aware of the influence incubation time might have on the phylogenetic composition of the isolate collection. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data can be found in the Supporting Information files. Competing interests: The authors have declared that no competing interests exist. However, sequencing can merely show which species are present at a specific time point. It provides useful information about the potential functions of a particular species or a community, but these still have to be tested in experimentally to verify actual functioning.
Also species traits that might be related to causes of species rarity, such as slow growth rates, the sensitivity to environmental conditions, competition or predation, have mostly been studied by analysing general changes in community composition or by other molecular techniques, such as stable isotope probing [ 4 , 5 ].
These potential traits will have to be verified in vitro. Therefore, cultivation is still required to study bacterial species in isolation and confirm potential traits. Present cultivation approaches are still largely untargeted without prior knowledge on the cultivable species except for the use of cultivation media selecting for or excluding a narrow range of species [ 6 ]. Hence, there is a clear need for more informed cultivation approaches to increase the chances of capturing bacteria of interest for subsequent studies both with respect to species abundance and to other traits, such as phylogeny.
In addition, it is still poorly investigated how the species that can be captured by cultivation, rank in abundance in the field community from which they have been isolated. The only studies that we are aware of, which looked at the abundance of cultivated species and rare species specifically, report contrasting results [ 8 , 9 ] that could partly be due to differences in cultivation techniques.
For example, there have been few, if any, studies on how different cultivation media might affect the abundance range of species captured. However, cultivation-independent studies give valuable information about some attributes of rare species that can be used to increase cultivation success. Therefore, the selection of small cells might increase the proportion of cultured rare species. In addition, it has been suggested that rare species might be low abundant in the community due to a poor competitive ability compared to species that are more abundant [ 11 ].
Therefore, the separation of single cells could favour the growth of rare species as it prevents direct competition.
In addition, some species that are rare in the field might belong to a temporally inactive seed-bank with dormancy as a life-history strategy to cope with unfavourable conditions [ 12 ]. Dormancy could lead to a poor cultivability of rare species in short-term cultivation approaches.
While a large proportion of dormant low abundant bacterial species has been shown to have the ability to become highly active and increase in abundance [ 13 ], it can be expected that re-activation from dormancy can cause these rare species to require longer incubation times for visible growth in cultivation approaches.
Moreover, rare species have often been assumed to be slow growing, which would also require a prolonged incubation time to increase the proportion of cultivated rare species.
However, in a follow-up study on bacterial isolates we did not find a difference in growth rate between rare or abundant species [ 14 ].
Not only is little known about the influence of cultivation media and incubation time on the cultivability of rare bacterial species, but also on species phylogeny. Extended incubation times have been indicated to lead to an increased cultivation of rarely isolated groups, such as Acidobacteria, Gemmatimonadetes or Planctomycetes [ 15 ]. However, it has seldom been systematically tested if phylogeny is related to incubation time.
Growth medium might also influence the phylogenetic composition of bacterial isolates. It has been suggested that nutrient-rich media can inhibit the growth of obligate slow-growing oligotrophic species [ 16 ]. Especially members of the Alphaproteobacteria have been indicated to grow preferentially on nutrient-poor media [ 17 ].
Moreover it has been suggested that autoclaving agar-containing media with high phosphate concentrations might inhibit the growth of some bacterial classes [ 18 , 19 ]. Examples of media that have been recommended for the isolation of soil bacteria are dilute tryptone soy broth or dilute nutrient broth DNB [ 20 , 21 ]. Different gelling agents, such as gellan gum, instead of agar, have been proven effective as well [ 20 ].
Attempts to mimic soil conditions have also led to the development of soil extract media, which yielded strains that could not grow on conventional media before [ 22 , 23 ]. Although these media have been used to successfully isolate as yet uncultivated species, remarkably little is known about the effect of different cultivation media on the general phylogeny of the isolated species.
The aim of the present study was to determine the relative abundance of cultivated bacterial species in field soil. We focused on the cultivation of bacterial species that are low abundant in soil, in order to enable further tests of their traits experimentally, e. Furthermore, we investigated whether long incubation times and different cultivation media would influence the proportion of cultivated rare species or the phylogenetic composition of the set of isolates. We found several rare species among our isolates, but at a lower proportion than would be expected from the proportion of rare bacterial species in the soil bacterial community.
Neither incubation time nor medium had an influence on the abundance of cultivated species, whereas incubation time influenced phylogenetic composition. The long-term biodiversity experiment has been installed in on an ex-arable field, abandoned from agriculture in Bacteria were isolated by a flow sorting in combination with different media. The following cultivation media were used: 0. Five well plates were prepared from each medium.
One day prior to cell sorting 5. Sonication was performed 2x 1 min to detach bacterial cells from soil particles.
After shaking again for 0. Louis, Missouri, USA. This method is known as fluorescence activated cell sorting FACS [ 26 ]. The size-selection was made to enhance the proportion of bacteria that are rare in field soil as it has been shown previously that the small-sized fraction of cells contains on average more rare species [ 10 ]. The sorted particles represented approximated 0. The plates were inspected every other day by eye for visible bacterial growth for 5 months.
Upon visible growth bacteria were subcultured by transferring them individually to fresh 0. This study was conducted before our follow-up study showed that there is no difference between rare and abundant taxa with respect to growth rates [ 14 ].
Therefore, rare species were still expected to require longer incubation times. We define cultivation success as the number of isolates that could be retrieved relative to the number of wells that were inoculated. After sequence quality trimming using the program Phred [ 27 ], sequences of a minimum length of 83 and a maximum length of bases were blasted against the greengenes and SILVA database using the SINA alignment service for phylogenetic identification [ 28 ] with a k-mer length of 10 bases.
The OTU reference table had been generated from a sequencing database containing sequences from seven soil replicates that were collected from the same site as the isolates, with — reads per sample [ 30 ]. The mean relative abundance of the OTU match in the sequencing database was taken as the relative abundance of the isolate in soil. For a detailed description of isolation of soil bacteria, identification of isolates, and estimation of relative abundance in the environment see [ 14 ]. The present study contains a subset of the isolates used in [ 14 ] as additional isolates were obtained by other methods that could not be directly compared with the flow sorting.
All statistical analyses were performed in R studio with R version 3. Abundance distributions between the field soil and unique isolated OTUs were compared with a Kolmogorov Smirnov-test. The abundance distributions of the different phylogenetic classes were tested in the same way.
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