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Many DNA isolation and protein expression protocols contain instructions to use a starter culture of E. coli that is then used to inoculate the main culture.
What are the advantages of using starter cultures compared to just let the bacteria grow in the same medium for a longer time? When should one use starter cultures and when can one safely skip that first culture?
Growth can be quite slow for some species under certain conditions when the concentration of cells is too low. Log-phase growth is powerful, and so one would like to keep cells in this state for the experiment at hand. Different genes are expressed then compared to a stationary phase.
In addition, you'd like your culture to out-compete a contaminant if there is one. That is more easily accomplished with a starter culture, which is then used to inoculate a larger culture for scale-up. Inoculating directing into the large-sized flask may allow your bacteria to enter a stationary phase, thus giving an opportunity for other species to out-compete your bacteria.
I agree that a starter culture would out-comete a contaminant (especially if there is no antibiotic in the media).
Another advantage of inoculating with starter culture is that your results concerning plasmid preps or preparing competent cells will be easily reproducible. By inoculating with a colony, the starter number of cells is different each time you do that, so you might not know how long your cells have to grow to reach a certain OD (e.g. for preparing competent cells) or you have to measure the OD before you isolate DNA to be sure that your yields are comparable with your previous ones.
What is the advantage of using starter cultures for growing bacteria? - Biology
Culture media is the food used to grow and control microbes.
- Culture media contains the nutrients needed to sustain a microbe.
- Culture media can vary in many ingredients allowing the media to select for or against microbes.
- Glucose or glycerol are often used as carbon sources, and ammonium salts or nitrates as inorganic nitrogen sources in culture media.
- culture: The process of growing a bacterial or other biological entity in an artificial medium.
- lysogeny broth: Lysogeny broth (LB) is a nutritionally-rich medium primarily used for the growth of bacteria.
Culture medium or growth medium is a liquid or gel designed to support the growth of microorganisms. There are different types of media suitable for growing different types of cells. Here, we will discuss microbiological cultures used for growing microbes, such as bacteria or yeast.
NUTRIENT BROTHS AND AGAR PLATES
These are the most common growth media, although specialized media are sometimes required for microorganism and cell culture growth. Some organisms, termed fastidious organisms, need specialized environments due to complex nutritional requirements. Viruses, for example, are obligate intracellular parasites and require a growth medium containing living cells. Many human microbial pathogens also require the use of human cells or cell lysates to grow on a media.
The most common growth media nutrient broths (liquid nutrient medium) or LB medium (Lysogeny Broth) are liquid. These are often mixed with agar and poured into Petri dishes to solidify. These agar plates provide a solid medium on which microbes may be cultured. They remain solid, as very few bacteria are able to decompose agar. Many microbes can also be grown in liquid cultures comprised of liquid nutrient media without agar.
Microbial pathogen growing on blood-agar plate: Red blood cells are used to make an agar plate. Different pathogens that can use red blood cells to grow are shown on these plates. On the left is staphylococcus and the right streptococcus.
DEFINED VS UNDEFINED MEDIA
This is an important distinction between growth media types. A defined medium will have known quantities of all ingredients. For microorganisms, it provides trace elements and vitamins required by the microbe and especially a defined carbon and nitrogen source. Glucose or glycerol are often used as carbon sources, and ammonium salts or nitrates as inorganic nitrogen sources. An undefined medium has some complex ingredients, such as yeast extract, which consists of a mixture of many, many chemical species in unknown proportions. Undefined media are sometimes chosen based on price and sometimes by necessity – some microorganisms have never been cultured on defined media.
There are many different types of media that can be used to grow specific microbes, and even promote certain cellular processes such as wort, the medium which is the growth media for the yeast that makes beer. Without wort in certain conditions, fermentation cannot occur and the beer will not contain alcohol or be carbonated (bubbly).
COMMON BROADLY-DEFINED CULTURE MEDIA
Nutrient media – A source of amino acids and nitrogen (e.g., beef, yeast extract). This is an undefined medium because the amino acid source contains a variety of compounds with the exact composition being unknown. These media contain all the elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory-culture collections.
Minimal media – Media that contains the minimum nutrients possible for colony growth, generally without the presence of amino acids, and are often used by microbiologists and geneticists to grow “wild type” microorganisms. These media can also be used to select for or against the growth of specific microbes. Usually a fair amount of information must be known about the microbe to determine its minimal media requirements.
Selective media – Used for the growth of only selected microorganisms. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing.
Differential media – Also known as indicator media, are used to distinguish one microorganism type from another growing on the same media. This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism. This type of media is used for the detection and identification of microorganisms.
These few examples of general media types provide some indication only there are a myriad of different types of media that can be used to grow and control microbes.
Classification of starter bacteria
The bacteria used in the manufacture of fermented dairy products are generally lactic acid bacteria (LAB) however, Propionibacterium shermanii and Bifidobacterium spp. which are not lactic acid bacteria, although Bifidobacterium species do produce lactic acid, are also used. In addition, other bacteria including Brevibacterium linens, responsible for the flavour of Limburger cheese and moulds (Penicillium species) are used in the manufacture of Camembert , Roquefort and Stilton cheeses.
There are currently sixteen genera in the LAB. Species from the Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus and Tetragenococcus genera are important in food fermentations and have recently been reviewed by the author for the Encylopedia of Food Microbiology, 2nd Edition. This section will review important properties of major genera used in dairy fermentations (Enterococcus, Lactobacillus, Lactococcus, Leuconostoc and Streptococcus).
These organisms are Gram-positive, catalase negative cocci that tend to form chains of varying length. They are normal inhabitants of the intestinal tract of man and other animals and are often used in microbiology as indicators of faecal contamination some species of the genus are pathogens. Apart from their ability to grow at 45°C, at pH 9.6, in high concentrations of salt, in high concentrations of bile salts, their general heat tolerance and their insensitivity to a range of antimicrobial agents they are superficially similar to lactococci. The biochemical identification key developed by Manero and Blanch (1999) is particularly helpful in identifing enterococci.
There are concerns about enterococci in foods partly because some are pathogens . However, it is their ability to exchange antibiotic resistance genes, particularly for glycopeptide antibiotics (vancomycin and teicoplanin), that perhaps raises most concern. Vancomycin is one of only a small number of antibiotics that may be effective against methicillin-resistant Staphylococcus aureus (MRSA).
Prior to recent taxonomic research, the Enterococcus species used as starters were classified as faecal streptococci and Group D streptococci.The posts in the forum on this group may also be of interest to readers.
Leuconostoc species are important flavour producers in some fermented dairy products. There is general agreement that two species, Leuconostoc mesenteroides subsp. cremoris and Lecon. lactis are important in starter cultures. Unlike lactococci, leuconostocs grow on Rogosa agar (see Billie et al., 1992 Mullan, 2000) and are hetrofermentative producing carbon dioxide from glucose and usually fructose. While the carbon dioxide production is undesirable in Cheddar cheese gas production is desirable in some varieties e.g. Emmental.
On microscopic examination, leuconostocs generally appear as Gram-positive cocci similar in size and shape (occur in pairs and in usually short chains) to lactococci. However, small rods can often be found and since leuconostocs grow on Rogosa agar, there can be a tendency to assume that these cultures are contaminated, with lactobacilli for example. Unlike lactococci, leuconostocs do not produce ammonia from arginine and produce the D isomer of lactic acid. With some exceptions leuconostocs only grow weakly in milk, and are not capable of reducing litmus before coagulation in litmus milk medium.
Isolation and identification of leuconostocs in starters is time consuming and laborious (see Billie et al., 1992) and the author has found that the use of Rogosa agar to obtain initial isolates helpful. Carbohydrate fermentation and identification of the lactic acid isomer are useful elements in an identification protocol.
Str. thermophilus is the only species of this genus found in starter cultures. This streptococcus is classified as a thermophile growing at 45°C, and higher, and is widely used in the manufacture of yoghurt and in Mozzarella, and in some other cheeses. More recently, probably since the mid-1990s-it has been used widely in the manufacture of Cheddar cheese. It is a component, along with lactococci, in some DVI / DVS cultures where it produces acid rapidly during scalding and may confer an additional measure of bacteriophage (phage) protection. Its incorporation in Cheddar-cultures also has the advantage of reducing the production costs of DVI/DVS cultures and controlling retail prices. Growth stops at around 15°C.
The slide shown in plate 1 was obtained by Gram-staining a yoghurt preparation the cocci are cells of Str. thermophilus and the rods are cells of Lb. delbrueckii subsp. bulgaricus. Like lactococci and many leuconostocs, strains of Str. thermophilus are catalase-negative coccus shaped and occur in pairs and chains. Generally, most strains produce long chains. L-lactic acid only is produced and carbon dioxide is not produced from glucose. Some strains produce urease and have the potential to produce CO2 from urea.
Since Str. thermophilus and Str. thermophilus-like organisms can grow in the regeneration section of pasteurisers high levels can occasionally occur in cheese. Urease-producing strains have the potential to cause openness in cheese (Mullan, 2000). Additionally the inability of many strains to metabolise galactose can result in cheese with significant concentrations of a fermentable carbohydrate that could be used by NSLAB for gas production. The potential involvement of Str. thermophilus should be considered during investigations of incidents of open texture or overt gas production in cheese. It is likely that the occasional problems of excessive early acidification encountered by Mozzarella manufacturers using extended production runs with pasteurised milk are due to NSLAB and in particular Str. thermophilus-like organisms that had grown to high cell densities in the regeneration section of pasteurisers.
Strains differ in their ability to utilise galactose. Use of non-galactose fermenting strains will result in high levels of this reducing sugar in products. Since galactose and other reducing sugars react with amino acids in the Maillard reaction it is usual to only select galactose-utilising strains to reduce the probability of undesirable colour changes occurring in heated products.
Str. salivarius, a streptococcus commonly found in saliva, has been shown by DNA: DNA hybridisation studies to be similar to Str. thermophilus (see references cited by Scheifer et al. (1991). Because of this, for some years Str. thermophilus was classified as a subspecies of Str. salivarius. However, it is now accepted that Str. thermophilus while similar, is sufficiently distinct to justify species designation.
Str. thermophilus is sensitive to low levels of salts and in particular to sodium chloride concentrations of around 2%. This sensitivity is important in using DVI / DVS cultures in Cheddar and similar cheeses. Cheese makers should understand that once the salt in moisture (S / M) concentration exceeds 2 - 3% lactose utilisation and acid production stops.
M17 medium (Terazghi and Sandine, 1975) widely used in studies with lactococci is not an ideal medium for the growth of some strains unless modified to reduce its glycerophosphate concentration. Note that many of the Str. thermophilus-like strains isolated from pasteurisers are much less sensitive to salt and normally grow satisfactorily on M17 agar.
This genus consists of a large group of Gram positive, catalase negative, rod-shaped bacteria. Some species are homofermentative while others are hetrofermentative. While some species produce mainly L-lactate from glucose, others produce D-lactate. Since some strains exhibit significant racemase activity, a racemase is an isomerase enzyme, D/L lactic acid is also produced. Strains may also exhibit coccoid morphology and this can lead, as discussed previously, to confusion with leuconostocs and perhaps even lactococci.
Lactobacilli are used as starters in the manufacture of yoghurt, and Mozzarella cheese. They are also used as starter adjuncts to promote faster ripening of Cheddar and similar cheeses, to reduce the incidence of bitterness and as probiotics in yoghurt type products. Note that there are several postings concerning the control of bitterness in Cheddar and Gouda cheeses in the discussion area.
Lb. delbrueckii subsp. bulgaricus is widely used along with Str. thermophilus as a starter in yoghurt manufacture. This subspecies is homofermentative, produces almost 2% w/v lactic acid in milk, has an optimum temperature of around 42°C and grows at temperatures of 45°C and higher. It will not grow in low concentrations of salt and is sensitive to bile salts.
Lb. acidophilus, which is normally present in the intestine, is generally not used as a starter it is widely used as a probiotic. This bacterium, is homofermentative, producing high concentrations of D-lactic acid in milk, has an optimum temperature of 37°C, and is relatively tolerant of oxygen, compared with Bifidobacterium species that are frequently used in conjunction with this organism. Little growth occurs at temperatures less than 20°C and most strains show no growth at 15°C. Because Lb. acidophilus produces D-lactate there have been some concerns about its use in infant nutrition. This aspect will be discussed further in the probiotics section.%
Lb. casei is also a normal inhabitant of the small intestine and is resistant to bile. It is used as a probiotic although it is found in some starter cultures and is commonly one of a number of non-starter lactic acid bacteria (NSLAB) found in Cheddar cheese. L-lactate is the main isomer of lactose produced although some strains produce small concentrations of D-lactate due to weak racemase activity. Rogosa agar is widely used as a general isolation medium for lactobacilli. Further information on enumeration is given in the article on probiotic bacteria.
Lb.helveticus is frequently used along with other thermophilic lactic acid bacteria in the manufacture of a range of fermented milk products including Emmental cheese, Mozzarella and yoghurt. One advantage of including this species along with Lb. delbrueckii subsp. bulgaricus is that Lb.helveticus utilises galactose and this can be useful if products free of reducing sugars are required. Since many strains have been shown to possess proline-iminopeptidase-like activity, Lb.helveticus has been used to produce modified 'Cheddar-type cheese' with some of the 'sweetness' characteristics of Swiss cheeses like Emmental. More recently, designated strains have been used as starter adjuncts to reduce bitterness in a range of cheeses, to improve flavour and/or to accelerate ripening. Bitterness is reduced due to peptidase action on starter-derived hydrophobic peptides. The species is homofermentative and produces high concentrations of D/L lactic acid in milk. Many strains grow at 45°C although lower temperatures 42-43°C generally give higher recoveries when enumerated using selective media such as Rogosa or modified MRS agars. Most strains show no or little growth at 15 °C (some atypical strains may take several weeks to grow at 15 °C or below). See the discussion area for further discussion on bitterness.
Originally, the bacteria in this group were classified as members of the genus Streptococcus and were designated as lactic streptococci. They were differentiated from other streptococci, some of which are pathogens, by their specific reaction with Group N antiserum and by their tolerance to temperature, salt and dyes (Jones, 1978). It is now known that serotyping lactic LAB has limited value in species differentiation strains of the same species may react with different sera and some strains may exhibit no group antigen (Schleifer and Kilpper-Balz, 1987). More information on why the lactic streptococci were reclassified is available here.
Differentiation of lactococci to species level
Lactococci can be differentiated to the species or biovariant level using the scheme developed for lactic streptococci-see above. Note that lactococci will not grow on Rogosa agar (Bille et al., 1992). Differential, but not selective, media are available and can be useful for quality control and strain isolation purposes. The medium, Reddys' Differential Agar, developed by Reddy et al. (1972) is still of value. This medium contains the differential ingredients lactose, calcium citrate, L-arginine and the pH indicator bromocresol purple. This indicator gives yellow and blue/purple colours under acid and alkaline conditions respectively.
Lc. lactis subsp. cremoris (shown in plate 2) gives yellow colonies due to acid production from lactose. Lc. lactis subsp. lactis while producing acid also produces ammonia from arginine. The ammonia neutralises the acid and eventually produces an alkaline reaction that results in blue/purple coloured colonies. Lc. lactis subsp. lactis biovar. diacetylactis also gives a blue/purple colony. Unlike Lc. lactis subsp. lactis, however, Lc. lactis subsp. lactis biovar. diacetylactis exhibits zones of clearing around colonies because of citrate utilisation. Because some strains of Lc. lactis subsp. lactis possess only weak arginase activity streaking techniques on an improved version of this medium may be helpful in identifying these strains (Mullan and Walker, 1979).
Advantages of Animal Cell Culture:
Ø Physio-chemical environment in the culture such as pH, temperature, osmolarity and level of dissolved gases can be precisely controlled in the in vitro system.
Ø Physiological conditions such as level of hormones and nutrients in the cell culture can be controlled.
Ø It is possible to control the micro-environment of the cells in the culture such as regulation of matrix, cell-cell interactions and cell substrate attachment.
Ø Cell culture techniques allow us to maintain the homogeneity of cells by the use of selective media.
Ø Cells in culture can be easily characterized by cytological or immune-staining techniques.
Ø Cells cultures can be stored in liquid nitrogen for very long time with a suitable cryopreservation medium such as DMSO.
Ø Cells in the culture can be easily quantified by different cell quantification techniques.
Ø The concentration (C) and time (T) dependent (C X T) effects of compounds such as pharmacologically active molecules, drugs or toxins can be easily studied by cell culture methods.
Ø The cell culture technique can be used for in vitro cytotoxicity studies to test the possible toxicity of compounds or drugs.
Ø More importantly, the uses of animals in scientific experiments (research) were significantly reduced with the invention of animal cell culture techniques.
Ø Cell culture can be used to produce monoclonal antibodies with hybridoma technology.
Ø Most of the molecular pathways that taking place inside a cell was elucidated by the use of cell culture techniques.
Ø With the invention of live cell imaging technique and fluorescent tagging methods, many physiological and molecular events in the cells can be visualized in a relatively inexpensive way through the use of in vitro cultured cells.
Ø Using molecular techniques, primary cells can be transformed and then it can be sub-cultured for unlimited passages.
Disadvantages of Animal Cell Culture
Ø Maintaining the sterile aseptic condition is the most difficult part of cell culture.
Ø Chances of chemical and microbial contamination are very high in in vitro methods.
Ø High possibility of cross contamination of different types of cells in culture.
Ø Experience and expertise are required for an effective maintenance most of the cells.
Ø The capital investment to set-up a cell culture facility is very high.
Ø Require through standardization of medium, concentration of nutrients and serum. All these vary with different types and origin of cells.
Ø Due to the rapid growth rate of cells artificial culture, there is a high chance of genetic variation within in a cell population.
Ø The high genomic variability can ultimately lead to heterogeneity of cells within the population and that cannot be easily distinguished.
Ø Identification of cell type is often difficult since in most of the cases the marker proteins will not express in ample quantity under in vitro conditions.
Ø The micro-environment in the culture vessel can induce many physical, chemical and physiological changes in the cells.
Ø Most of the primary cell in culture will only have limited number of passages.
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Food bacteria-spice survey shows why some cultures like it hot
Fans of hot, spicy cuisine can thank nasty bacteria and other foodborne pathogens for the recipes that come -- not so coincidentally -- from countries with hot climates. Humans' use of antimicrobial spices developed in parallel with food-spoilage microorganisms, Cornell University biologists have demonstrated in a international survey of spice use in cooking.
The same chemical compounds that protect the spiciest spice plants from their natural enemies are at work today in foods from parts of the world where -- before refrigeration -- food-spoilage microbes were an even more serious threat to human health and survival than they are today, Jennifer Billing and Paul W. Sherman report in the March 1998 issue of the journal Quarterly Review of Biology.
"The proximate reason for spice use obviously is to enhance food palatability," says Sherman, an evolutionary biologist and professor of neurobiology and behavior at Cornell. "But why do spices taste good? Traits that are beneficial are transmitted both culturally and genetically, and that includes taste receptors in our mouths and our taste for certain flavors. People who enjoyed food with antibacterial spices probably were healthier, especially in hot climates. They lived longer and left more offspring. And they taught their offspring and others: 'This is how to cook a mastodon.' We believe the ultimate reason for using spices is to kill food-borne bacteria and fungi."
Sherman credits Billing, a Cornell undergraduate student of biology at the time of the research, with compiling many of the data required to make the microbe-spice connection: More than 4,570 recipes from 93 cookbooks representing traditional, meat-based cuisines of 36 countries the temperature and precipitation levels of each country the horticultural ranges of 43 spice plants and the antibacterial properties of each spice.
Garlic, onion, allspice and oregano, for example, were found to be the best all-around bacteria killers (they kill everything), followed by thyme, cinnamon, tarragon and cumin (any of which kill up to 80 percent of bacteria). Capsicums, including chilies and other hot peppers, are in the middle of the antimicrobial pack (killing or inhibiting up to 75 percent of bacteria), while pepper of the white or black variety inhibits 25 percent of bacteria, as do ginger, anise seed, celery seed and the juices of lemons and limes.
The Cornell researchers report in the article, "Countries with hotter climates used spices more frequently than countries with cooler climates. Indeed, in hot countries nearly every meat-based recipe calls for at least one spice, and most include many spices, especially the potent spices, whereas in cooler counties substantial fractions of dishes are prepared without spices, or with just a few." As a result, the estimated fraction of food-spoilage bacteria inhibited by the spices in each recipe is greater in hot than in cold climates.
Accordingly, countries like Thailand, the Philippines, India and Malaysia are at the top of the hot climate-hot food list, while Sweden, Finland and Norway are at the bottom. The United States and China are somewhere in the middle, although the Cornell researchers studied these two countries' cuisines by region and found significant latitude-related correlations. Which helps explain why crawfish etoufŽe is spicier than New England clam chowder.
The biologists did consider several alternative explanations for spice use and discounted all but one. The problem with the "eat-to-sweat" hypothesis -- that people in steamy places eat spicy food to cool down with perspiration -- is that not all spices make people sweat, Sherman says, "and there are better ways to cool down -- like moving into the shade." The idea that people use spices to disguise the taste of spoiled food, he says, "ignores the health dangers of ingesting spoiled food." And people probably aren't eating spices for their nutritive value, the biologist says, because the same macronutrients are available in similar amounts in common vegetables, which are eaten in much greater quantities.
However the micronutrient hypothesis -- that spices provide trace amounts of anti-oxidants or other chemicals to aid digestion -- could be true and still not exclude the antimicrobial explanation, Sherman says. However, this hypothesis does not explain why people in hot climates need more micro-nutrients, he adds. The antimicrobial hypothesis does explain this.
The study of Darwinian gastronomy is a bit of a stretch for an evolutionary biologist like Sherman, who normally focuses his research on the role of natural selection in animal social behavior and is best known for his studies of one of nature's most social (and unusual-looking) creatures, the naked mole-rat (Heterocephalus glaber) of Africa. But eating is definitely one of the more social behavior of Homo sapienss, he maintains, and it's a good way to see the interaction between cultural evolution and biological function. "I believe that recipes are a record of the history of the coevolutionary race between us and our parasites. The microbes are competing with us for the same food," Sherman says. "Everything we do with food -- drying, cooking, smoking, salting or adding spices -- is an attempt to keep from being poisoned by our microscopic competitors. They're constantly mutating and evolving to stay ahead of us. One way we reduce food-borne illnesses is to add another spice to the recipe. Of course that makes the food taste different, and the people who learn to like the new taste are healthier for it."
For biology student Billing, the spice research for a senior honors thesis took her to an unfamiliar field, food science, and to the Cornell University School of Hotel Administration, where the library contains one of the world's largest collections of cookbooks. Now that the bacteria-spice connection is revealed, librarians everywhere may want to cross-index cookbooks under "food safety." And spice racks may start appearing in pharmacies.
Top 30 Spices with Antimicrobial Properties
(Listed from greatest to least inhibition of food-spoilage bacteria)
Source: "Antimicrobial Functions of Spices: Why Some Like It Hot," Jennifer Billing and Paul W. Sherman, The Quarterly Review of Biology, Vol. 73, No.1, March 1998
What Is a Bacterial Culture? (with pictures)
A bacterial culture is a cultivated colony of bacteria grown in a lab for a variety of purposes, ranging from patient diagnosis to scientific research. Cultures can take hours or days to grow and may require special care, as some bacteria are very finicky about their environment. Lab technicians typically follow a specific set of procedures to standardize the culturing process and increase the chances of success.
Successful cultures require a good sample. Doctors can use swabs to collect specimens from the site of an infection, or they can submit blood, urine, and other fluids for culture. In the case of environmental research, samples of soil, infected tissue from plants, and water can be useful for a bacterial culture. The sample must be stored in optimal conditions to ensure survival of the bacteria until they reach the lab.
There are several ways to set up a bacterial culture. One of the most common is the petri dish. The technician will prepare a shallow dish filled with gel, usually made from agar, a component of seaweed. The gel contains nutrients to support the bacteria after they are introduced. Another option is a nutrient broth, where the bacteria will be suspended in liquid. In both cases, the bacterial culture goes into an incubator to promote growth, and the technician will periodically inspect it.
As bacteria start to grow, they can cause visible changes in the culture medium. Streaks and dots may appear, and the colony can turn a variety of colors as it spreads. The technician can use a microscope to examine the bacteria and learn more about them with testing like gram staining. Technicians can also add antibiotics to the culture to test antibiotic sensitivity. If the bacteria die off, it means the drugs are working, and if the bacteria keep growing, it means they are resistant to that particular medication.
Doctors can order a bacterial culture if they believe a patient has an infection and they want to confirm the presence of disease and determine the most appropriate medication to treat it. Researchers grow bacteria to identify useful compounds, learn more about their role in the environment, and extract bacterial toxins for research. Labs must observe careful bacterial culture protocols to limit contamination, infection, and other concerns. Most are subject to inspection by regulatory officials who will make sure appropriate safety measures are being exercised.
Ever since she began contributing to the site several years ago, Mary has embraced the exciting challenge of being a InfoBloom researcher and writer. Mary has a liberal arts degree from Goddard College and spends her free time reading, cooking, and exploring the great outdoors.
Ever since she began contributing to the site several years ago, Mary has embraced the exciting challenge of being a InfoBloom researcher and writer. Mary has a liberal arts degree from Goddard College and spends her free time reading, cooking, and exploring the great outdoors.
The FISH Technique
FISH is a hybridization technology which allows the labeling of target RNAs with a fluorescent probe.
Describe how fluorescent in situ hybridization (FISH) is used in clinical and biomedical studies to detect and localize the presence or absence of specific DNA sequences and to identify pathogens
- FISH can be used in a clinical setting to identify pathogens or DNA / RNA targets of interest.
- FISH is used to detect and localize the presence or absence of specific DNA or RNA sequences in tissue or cells.
- FISH can also be used to compare the genomes of two biological species, such as in ecological studies, where a bacteria may not be culturable, it can be identified using FISH.
- fluorescence: The emission of light (or other electromagnetic radiation) by a material when stimulated by the absorption of radiation or of a subatomic particle
- hybridize: To combine complementary subunits of multiple biological macromolecules.
- FISH: Fluorescence in situ hybridization is a cytogenetic technique used to detect and localize thespecific DNA or RNA sequences.
FISH (fluorescence in situ hybridization ) is a cytogenetic technique developed by biomedical researchers in the early 1980s. It is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes bind to those targets that show a high degree of sequence complementarity. FISH can be used to detect RNA or DNA sequences of interest. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets, including mRNAs, in cells. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.
Central to FISH are the use of probes. The probe must be large enough to hybridize specifically with its target but not so large as to impede the hybridization process. They are anti-sense to the target mRNA or DNA of interest, thus they hybridize to targets. The probe can be tagged directly with fluorophores, or with targets for flourescently labelled antibodies or other substrates. Different types of tags can be used, therefore different targets can be detected in the same sample simultaneously (multi-colour FISH). Tagging can be done in various ways, such as nick translation, or PCR using tagged nucleotides. Probes can vary in length from 20 to 30 nucleotides to much longer sequences.
Dual label FISH image: Here is an example of FISH being used to differentiate Bifidobacteria (red) and other bacteria (green)
FISH is often used in clinical studies. If a patient is infected with a suspected pathogen, bacteria from the patient’s tissues or fluids, are typically grown on agar to determine the identity of the pathogen. Many bacteria, however, even well-known species, do not grow well under laboratory conditions. FISH can be used to directly detect the presence of the suspect on small samples of the patient’s tissue. FISH can also be used to compare the genomes of two biological species, to deduce evolutionary relationships. A similar hybridization technique is called a zoo blot. Bacterial FISH probes are often primers for the 16s rRNA region. FISH is widely used in the field of microbial ecology, to identify microorganisms. Biofilms, for example, are composed of complex (often) multi-species bacterial organizations. Preparing DNA probes for one species and performing FISH with this probe allows one to visualize the distribution of this specific species within the biofilm. Preparing probes (in two different colors) for two species allows to visualize/study co-localization of these two species in the biofilm, and can be useful in determining the fine architecture of the biofilm.
There are a few different ways to prepare brine for fermenting vegetables, including a method for fermenting without salt. Choose the process that works best for you from the following choices:
Method #1: Salt-only Vegetable Fermentation
Historically, salt was used to preserve foods before refrigeration. We recommend salt-only fermented vegetables at CFH, for many reasons:
- Salt pulls out the moisture in food, denying bacteria the aqueous solution they need to live and grow.
- Salt allows the natural bacteria that exist on the vegetables to do the fermenting. Only the desired salt-tolerant Lactobacilli strains will live and propagate.
- By suppressing the growth of other bacteria and mold, salt provides a slower fermentation process that is perfect for cultured vegetables that are to be stored for longer periods of time.
- Salt hardens the pectins in the vegetables, leaving them crunchy and enhancing the flavor.
- Use 1-3 tablespoons of our authentic, finely-ground Celtic Sea Salt per quart of water to prepare brine for fermenting vegetables.
Method #2: Salt-free Vegetable Fermentation
- Salt-free ferments, while often more bio-diverse, can result in mushy vegetables and mold.
- For a salt-free ferment celery juice or seaweed may be substituted, but they will not prevent a mushy texture.
- Some freeze-dried starter cultures may be used on their own, without salt (see Method #3, below). Always follow the instructions included with the freeze-dried starter culture, for best results.
- This salt-free sauerkraut recipe uses herbal seeds in place of salt.
Method #3: Salt Plus Starter Cultures
Using some form of bacterial starter is said to speed up the vegetable fermentation process. While we recommend a salt-only ferment for vegetables, the following starter cultures may be used in addition to salt, if desired.
- Whey is dairy-based, so may not work for everyone. Make sure the whey is properly strained and fresh-tasting, as it will lend its flavor to the batch. Add salt along with the whey for flavor and to keep the vegetables crunchy.
- Freeze-Dried Starter Cultures: When using a freeze-dried starter culture, follow the instructions included with that culture, for best results.
If you prefer to use a freeze-dried starter culture, choose from our high-quality Vegetable Starter Cultures:
- Brine from a Previous Ferment: The fermented vegetable juice from a previous batch can be added to a new batch as a starter. Add about ¼ cup brine per quart of vegetables.
- Other Fermented Liquids: Finished, unflavored water kefir or kombucha may be used as a starter culture for fermenting vegetables. Add about ¼ cup liquid per quart of vegetables.
Where Do the Microbial Species in Sourdough Starters Come From?
In a January 2020 study, Reese et al. sought to determine the source of microbes by sequencing the bacteria and fungi in sourdough starters prepared by 18 professional bakers using a standardized recipe and ingredients. The microbes in the starters were compared to those on the bakers&rsquo hands and in the ingredients. M icrobes previously documented in sourdough starters were also found in the professionals&rsquo starters: the most common orders were Saccharomycetales for yeasts and Lactobacillales for bacteria. The microbial communities found in the starters were overall most similar to that found in the flour therefore, most of the bacteria and yeast arrive with the flour.
Where should I buy my starter culture from?
For starter cultures, simply purchase plain yogurt that states ‘live’ or ‘active’ cultures on its labels. Avoid fruit or frozen yogurts as they contain undesirable microorganisms as well as sugar. These undesirable microorganisms may out-compete the desirable bacteria.
You can safely use cow’s milk yogurt to inoculate goat milk and vice-a-versa. What is important is ‘live’ and ‘active’ strains.
If you use store-bought yogurt for starter, make sure you use freshly opened container to ensure that you only inoculate with desirable bacteria. Partially used, opened container left open on the breakfast table or that has been eaten with a not-so-clean spoon is a strict no-no when making yogurt.
Choose a brand of yogurt you like. Some yogurts are tangier, others are a bit mellow. If you use tart and tangy store-bought yogurt, then your batch will be tangy too. Also, make it a point to check the expiration date of your yogurt container because even an unopened container can become tangy over time.
Promptly freeze unwanted culture and use it up within a few months. Most remain viable for up to 6 months. In the case of dried cultures, more culture may not necessarily mean faster fermentation. In fact the opposite is more likely as the milk may not contain enough lactose to feed all of the bacteria. They may even die off before they coagulate the milk.