Cell Culture Techniques |Biotechnify|BTF

What is Cell Culture? 

Cell culture is the process by which cells are grown under controlled conditions, generally outside their natural environment. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions.

Essentially , cell culture involves the distribution of cells in an artificial environment (in vitro) which is composed of the necessary nutrients, ideal temperature, gases, pH and humidity to allow the cells to grow and proliferate.

• In vivo - When the study involves living biological entities within the organism.

• In vitro - When the study is conducted using biological entities (cells, tissue etc) that has been isolated from their natural biological environment. E.g. tissue or cells isolated from the liver or kidney. 

Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established.

What is Cell Culture Techniques ?

In cell culture techniques, cells (or tissues) are removed from a plant or an animal and introduced into a new, artificial environment that can support their proliferation (survival and growth). Some of the requirements of such an environment for the proliferation of the cells include: A substrate (source of nutrition).

Cell culture techniques play a key role in the development of new anticancer drugs by imposing additional constraints on those of receptor interaction alone, such as drug uptake and efflux, interaction with other cellular receptors, and cellular metabolism

Primary culture :

Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). At this stage, the cells have to be subcultured (i.e., passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth

Cell Culture Protocol :

Cell culture protocols are meant to ensure that culture procedures are carried out to the required standards. This is not only meant to prevent the contamination of the cells, but to also ensure that the researchers themselves are protected from any form of contamination.

However, the nature of the work is expected to conform to the appropriate ethical guidelines. Therefore, before anything else, it is essential to ensure that the entire procedure conforms with both medical-ethical and animal- experiment guidelines. This is because going against such legislation and guidelines can result in heavy penalties and even shutting down of the laboratory.

Before start , carry out the following procedure:

Ensure that the working are is sanitized (using 70 percent ethanol)Always use a new pair of gloves. If a pair of gloves has to be used for another cell culture procedure, they should be sanitized using 70 percent ethanol and allowed to air dry.

Any equipment that had been taken out of the cabinet should also be sanitized to prevent any contamination such equipment as pipette, glass jars and plastics to be used for the procedure should be autoclaved

Although there are a wide range of culture media for cells, it is important to keep in mind that cell cultures, and particularly primary cell cultures are easily prone to contamination in addition to the risk of containing undetected viruses. For this reason, all material should be handled as potentially infectious in order to avoid any infections.

Protocols for cell culture preparation :

Always check the information on the container to ensure that the medium is appropriate for the cell to be cultured,

Once prepared, the cell culture should be maintained under the recommended temperature range,

Monitor the culture every 30- 48 hours and check for confluency (when cells completely cover the surface of the culture) - However, this is largely dependent on the type of cells.

Once the procedure is completed and the cells have been analyzed, the culture should be appropriately discarded. Here, it is important to take a lot of caution given that by this time, cells have already proliferated and increased in numbers. Moreover, there are high chances that the specimen has been contaminated, which increase the risks of causing infections to the researcher if not handled appropriately.

What is Cell Culture used for? 

Cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the effects of drugs and toxic compounds on the cells, and mutagenesis and carcinogenesis.

What is Cell Passaging? 

Subculturing, also referred to as passaging cells, is the removal of the medium and transfer of cells from a previous culture into fresh growth medium, a procedure that enables the further propagation of the cell line or cell strain.

Applications of Cell Culture :

Cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the effects of drugs and toxic compounds on the cells, and mutagenesis and carcinogenesis. It is also used in drug screening and development, and large scale manufacturing of biological compounds (e.g., vaccines, therapeutic proteins). The major advantage of using cell culture for any of these applications is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells.

Conclusion :

In conclusion, cell culture is an indispensable tool in modern day medicine and its applications are innumerable in diagnosis of human infection. Cell culture methods are unbiased to some extent and only limited by the ability of the virus to grow in a particular cell line.

Isolation Techniques - short explanation | notes on isolation techniques

 Isolation Techniques - short explanation | notes on isolation techniques

Isolation Techniques 

1. the process of separating, or the state of being alone.

2. the physiologic separation of a part, as by tissue culture or by interposition of inert material.

3. the extraction and purification of a chemical substance of unknown structure from a natural source.

4. the separation of infected individuals from those uninfected for the period of communicability of a particular disease; see also quarantine.

5. the separation of an individual with a radioactive implant from others to prevent unnecessary exposure to radioactivity.

Definition :

Isolation refers to the precautions that are taken in the hospital to prevent the spread of an infectious agent from an infected or colonized patient to susceptible persons.

In microbiology, the term isolation refers to the separation of a strain from a natural, mixed population of living microbes, as present in the environment, for example in water or soil flora, or from living beings with skin flora, oral flora or gut flora, in order to identify the microbe(s) of interest.

History :

The laboratory techniques of isolating microbes first developed during the 19th century in the field of bacteriology and parasitology using light microscopy. Proper isolation techniques of virology did not exist prior to the 20th century. The methods of microbial isolation have drastically changed over the past 50 years, from a labor perspective with increasing mechanization, and in regard to the technologies involved, and with it speed and accuracy.

Purpose

Isolation practices are designed to minimize the transmission of infection in the hospital, using current understanding of the way infections can transmit. Isolation should be done in a user friendly, well-accepted, inexpensive way that interferes as little as possible with patient care, minimizes patient discomfort, and avoids unnecessary use.

Precautions Types

There are three types of transmission-based precautions--contact, droplet, and airborne - the type used depends on the mode of transmission of a specific disease.

Precautions :

The type of precautions used should be viewed as a flexible scale that may range from the least to the most demanding methods of prevention. These methods should always take into account that differences exist in the way that diseases are spread. Recognition and understanding of these differences will avoid use of insufficient or unnecessary interventions.

Standard precautions :

Standard Precautions define all the steps that should be taken to prevent spread of infection from person to person when there is an anticipated contact with:

Blood

Body fluids

Secretions, such as phlegm

Excretions, such as urine and feces (not including sweat) whether or not they contain visible blood

Nonintact skin, such as an open wound

Mucous membranes, such as the mouth cavity.

Standard Precautions includes the use of one or combinations of the following practices. The level of use will always depend on the anticipated contact with the patient:

Handwashing, the most important infection control method

Use of latex or other protective gloves

Masks, eye protection and/or face shield

Gowns

Proper handling of soiled patient care equipment

Proper environmental cleaning

Minimal handling of soiled linen

Proper disposal of needles and other sharp equipment such as scalpels

Placement in a private room for patients who cannot maintain appropriate cleanliness or contain body fluids.

Transmission based precautions :

Transmission Based Precautions may be needed in addition to Standard Precautions for selected patients who are known or suspected to harbor certain infections. These precautions are divided into three categories that reflect the differences in the way infections are transmitted. Some diseases may require more than one isolation category.

AIRBORNE PRECAUTIONS. Airborne Precautions prevent diseases that are transmitted by minute particles called droplet nuclei or contaminated dust particles. These particles, because of their size, can remain suspended in the air for long periods of time; even after the infected person has left the room. Some examples of diseases requiring these precautions are tuberculosis, measles, and chickenpox.

Following isolation methods are employed to isolate microbes from mixed cultures:

1. Streaking

2. Plating

3. Dilution

4. Enriched procedure, and

5. Single cell technique.

Description :

Isolation practices can include placement in a private room or with a select roommate, the use of protective barriers such as masks, gowns and gloves, a special emphasis on handwashing (which is always very important), and special handling of contaminated articles. Because of the differences among infectious diseases, more than one of these precautions may be necessary to prevent spread of some diseases but may not be necessary for others.The Centers for Disease Control and Prevention (CDC) and the Hospital Infection Control Practice Advisory Committee (HICPAC) have led the way in defining the guidelines for hospital-based infection precautions. The most current system recommended for use in hospitals consists of two levels of precautions. The first level is Standard Precautions which apply to all patients at all times because signs and symptoms of infection are not always obvious and therefore may unknowingly pose a risk for a susceptible person. The second level is known as Transmission-Based Precautions which are intended for individuals who have a known or suspected infection with certain organisms.

Frequently, patients are admitted to the hospital without a definite diagnosis, but with clues to suggest an infection. These patients should be isolated with the appropriate precautions until a definite diagnosis is made.

Notes :

It is absolutely essential that you sterilize your loop between each streaking, either by using the incinerator or by obtaining a new sterile plastic loop. This is the most common mistake students make.

Don’t leave your plate open too long or extra bacteria from the environment will fall into your plate.

Do not be disappointed if you do not get isolated colonies on your first try. This is a difficult procedure.