Bacteriology

Bacterial Culture Technique

Microbiological cultures, also known as Microbial cultureis the technique of generating microbial species by the process of letting them reproduce in a...

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This article writter by MN Editors on February 15, 2022

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Bacterial Culture Technique
Bacterial Culture Technique

Microbiological cultures, also known as Microbial culture is the technique of generating microbial species by the process of letting them reproduce in a conditions in a monitored laboratory conditions. Microbiological cultures are fundamental and essential diagnostic methods that are employed as research tools within molecular biology. The term “culture” could be used to describe the microorganisms that are being grown.

Microbial cultures can be utilized to identify the kind of organism, the amount in the sample that is being examined or both. It is among the main diagnostic methods in microbiology. They are used as tool to discover the source of infection by allowing the organism to grow in a specific medium. For instance the throat culture can be made by scraping off the lining of the tissue at an area behind the throat, and then blotting the tissue into the medium in order to detect harmful microorganisms like Streptococcus pyogeneswhich is the causing agent responsible for the condition known as strep throat. Additionally, the term “culture” is used more often to mean “selectively developing” the specific type of microorganism that is in the laboratory.

It is usually necessary to isolate a purified microorganism culture. Pure (or an axenic) cultivar is group of multicellular organisms or cells that are growing in the absence of other species or kinds. Pure cultures may come from one single cell or organism and, in this case, these cells have genetic counterparts of other. In order to gel the microbial growth the medium of Agarose Gel (agar) is employed. Agar is a gelatinous material that is derived from seaweed. An alternative to agar that is inexpensive is guar gum. It can be used for removal and maintaining thermophiles.

The methods used to cultivate cultures are essential for a laboratory of microbiology. Different methods of culture are carried out to:

  • Remove bacteria in pure culture, and then identify them by performing a variety of tests.
  • Show the biochemical, antigenic and other phenotypic as well as genomic characteristics of the isolated colonies.
  • The isolate should be able to demonstrate susceptibility microorganisms to antibiotics and bacteriophages Bacteriocins, etc.
  • Create antigens to be used in a variety of ways.
  • Keep the stock culture.

Bacterial Culture Technique

Different methods are employed for cultivating bacteria. They include (a) streak culture, (b) lawn culture, (c) pour-plate culture, (d ) stroke culture, (e) stab culture, and ( f ) liquid culture.

Bacterial Culture Technique
Bacterial Culture Technique | Image Source: brainkart.com

1. Streak Culture

The streak culture method is by far the best method to obtain discrete colonies of bacteria. It is accomplished by streaking the top of the media plate with the loop of nichrome or platinum that is 2-4 millimeters in diameter. In this technique, the inoculum in a loop is put in the edges on the surface of the plate. The inoculum then is distributed using the loop until it covers about one-fourth the size of the plate by very close strokes. From the inoculum that was originally created, the inoculum is spread out thinly on the plate, streaking an in-line loop. 

The loop is heated before being cools in between the streaks in order to create isolated colonies. The inoculated plate is kept at 37°C overnight to show the colonies. Confluent growth begins within the inoculum that is primary however, it becomes thinner. Separated colonies are observed in the last stripes of the inoculum. The isolated colonies that are obtained using this method can be extremely useful to examine the various characteristics of bacteria. This is by far the most effective method to obtain discrete colonies of bacteria.

2. Lawn Culture

The lawn culture creates an even layer of bacteria on a medium. It is done by saturating the surface of the dish with liquid or suspension, piping off the excess inoculum and then incubating the plate for several hours at 37°C. The cul-ture plate can be inoculated with an sterile swab that has been that is soaked in liquid bacterial cultures or suspension and incubating over night to observe the colonies of bacterium.

3. Pour-Plate Culture

The pour-plate is used to estimate the number of viable organisms present in liquids such as urine or water. It’s used to measure the presence of bacteria in urine and to determine the number of viable bacteria in the suspension. This procedure is carried out in tubes, each of which contains 15mL of molten agar. The molten agar inside the tubes is then allowed for cooling in a bath of water at 45°C. 

The inoculum that is to be test is reduced by sequential diluting. One mL of the diluted inoculum will be added into each tube of the molten agar , and blended well. The contents of the tubes are then poured into sterilized Petri dishes and left to settle. After overnight incubation in this Petri dishes at 37 degrees Celsius the colonies appear to be spread throughout the entire deepest part of the medium which can be counted with an instrument called a colony counter.

4. Stroke Culture

Stroke culture is the pure strain of bacteria that can carry the slide agglutination as well as other tests for diagnosing. It is conducted in tubes, which typically contain nutrients Agar slopes.

5. Stab Culture

Stab culture is made by stapling the medium into tubes using a long straight wire, and then incubating it at 37°C.

6. Liquid Culture

Liquid culture is made in a liquid medium enclosed in flasks, tubes or bottles. Inoculation of the medium takes place through the contact of an charged loop or adding the inoculum via pipettes or syringes, and then incubating at 37 degrees Celsius, then subculture onto solid media to identify the final culture.

The main drawback in liquid-based culture is that it doesn’t provide a pure culture of bacteria, and the growth of the bacteria does not display distinctive characteristics.

Anaerobic Culture

Obligate anaerobes, also known as obligate anaerobes are able to live in presence of oxygen. These anaerobes die by exposure to air for as little as 5 minutes. Certain anaer-obes can tolerate tiny levels of oxygen. Facultative anaerobes are the anaerobes which grow in the absence of oxygen.

Anaerobic bacterial cultures are a technique used to create anaerobes from a specimen taken from a clinic. The identification and culture of anaerobes is crucial for starting the appropriate treatment.

In the event of a failure to act, it can have severe consequences, like an organ failure, amputations sepsis or meningitis and even death.

Specimen Collection for Anaerobic Culture

Anaerobic culture is often based on a variety of specimens. culture are:

  • Blood, bone marrow and bile cerebrospinal fluid; direct aspirate of the lung and tissue biopsy taken from a normally safe area;
  • A fluid aspirated from a non-sterile location, such as an joint
  • Pus samples of dental abscesses or burn wounds and pelvic or abdominal abscess; and
  • The specimens are from gunshot, knife as well as surgical cuts.

The collection of a specimen that is free of contamination and shielding the specimen from exposure to oxygen in the process of collection is the primary goal of anaerobic cultures. The samples must be taken from a safe place without contaminating the specimen with bacteria from the surrounding mucous membrane, skin or even tissue.

Abscesses, or fluids, are generally taken care of using a sterilized needle and then sealed to stop the entry of air. Tissue samples are placed in bags that have been degassed and sealed, or in an open screw top vial, which could contain oxygen-free culture medium that has been tightly sealed. The samples should be placed as quickly as they can onto the culture medium to isolate bacteria.

Importance of Bacterial Culture Technique

There are a variety of reasons it might be beneficial or beneficial to cultivate bacteria. We will examine a few of the most common reasons.

1. Diagnose infection

Despite the time it takes to identify and isolate the bacterial species in an contaminated sample, bacterial cultures remains a crucial diagnostic tool.12 Although PCR could quickly detect any particular pathogen, the process of identifying the source can confirm that the pathogen remains alive and alerting scientists to possible transmission risks and providing how to treat. Also, the bacterium can be further examined for additional specific information such as sensitivity to antibiotics as well as directing treatment decisions. The strains could also be put in the future for analysis, such as for monitoring of diseases.

2. Genetic manipulation

It could be beneficial to alter the genome of bacteria for a variety of reasons. For example, seeking to better understand the fundamental biologyof the organism, to lessen its impact in the creation of vaccine strains, or to increase the production of proteins, and make a reference strain that has the ability to detect a marker to name only a few. When it comes to mutating, deleting or introducing genetic material there is a need to establish the strain of interest prior to or after the process of genetic engineering.

3. Epidemiological study

The cultivation and identification of bacteria can be crucial for epidemiological studies.14 This allows scientists to examine how the bacterial population changes in time. This can help inform vaccine, therapeutic and diagnostic designs and updates, and also examine transmission events that can be used to inform public health policy and guidance. This Gonococcal Surveillance project (GISP) is a good example of a project that studies the strains of antibiotic resistance and assists in forming the recommendations for drug therapy. It is run by the Centers for Disease Control and Prevention (CDC) also oversees its active Bacterial Core monitoring (ABCs) system that provides the surveillance of populations and laboratories of invasive pathogens affecting bacterial populations that are of concern to public health.

4. Scale up to enable omics studies

Although the sequencing of DNA and RNA is feasible using tiny quantities of genetic material even at the single cell level, for a lot of studies Next-Generation Sequencing (NGS) is still carried out using bacteria. As so, bacteria usually require a culture before DNA extraction or DNA or extraction.15 If you’re looking for particular strains (unlike microbiome research which may include the mix of) the strain will originate from a pure culture.

5. Develop vaccines and therapeutics

To fight the bacterial pathogen that you want to fight generally, you must be able of cultivating the pathogen as well. When developing vaccines, it is possible to isolate strains of bacteria to learn their genomes to enhance their genes or alter the genes. Additionally, to evaluate potential therapies or vaccines It is typically required to conduct challenge experiments17 that test subjects with the pathogen to test whether the treatment is effective. In order to do this the bacterium is typically grown and, when in the form of a specific challenge model, is enumerated in order to assess the amount of dose that subjects are given.

6. Food and beverage production

Bacteria are a crucial component in the creation of numerous foods. They are divided into starter and probiotic culture.

Probiotics are usually cultivated to increase their health benefits,18 usually through our gut microbiome. Probiotics can contain many different species of bacterial Lactobacillus, Bifidobacterium and Bifidobacterium are the most popular selections to cultivate.

Starter culture, however, are usually used as an element of a process of food production to improve flavor nutrition value, texture or enhance preservation. For instance, sourdough breads salami, 19 pepperoni, and dried Ham. The lactic acid bacteria (LAB) are often found in the starter cultures. Certain drinks and foods might, however, be between the two camps like yogurt, and the ever-growing popularity of kimchi20 as well as Kombucha. These drinks are consumed due to their taste as well as their probiotic benefits.

No matter what purpose the culture was created for maintaining a healthy environment that is free of contaminants is crucial for efficient production and security.

7. Detecting food contaminants

Although some bacteria are useful in the food industry however, they could also be a contamination and could be able to cause serious foodborne illness. The most common causes are Salmonella as well as., Listeria monocytogenes, Campylobacter jejuni as well as E. coli. It is crucial that researchers are able to cultivate any potentially harmful bacteria in food samples, even if they’re present in small quantities.

References

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Microbiology Notes is an educational niche blog related to microbiology (bacteriology, virology, parasitology, mycology, immunology, molecular biology, biochemistry, etc.) and different branches of biology.

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