Fermentation |Biotechnify |BTF

 

Fermentation : Definition & Introduction 

The chemical breakdown of a substance by bacteria, yeasts, or other microorganisms, typically involving effervescence and the giving off of heat.

"the fermentation of organic matter by microorganisms in the gut"

Fermentation is a metabolic process that produces chemical changes in organic substrates through the action of enzymes. In biochemistry, it is narrowly defined as the extraction of energy from carbohydrates in the absence of oxygen.

The science of fermentation is known as zymology.

In microorganisms, fermentation is the primary means of producing adenosine triphosphate (ATP) by the degradation of organic nutrients anaerobically.[2] Humans have used fermentation to produce foodstuffs and beverages since the Neolithic age. For example, fermentation is used for preservation in a process that produces lactic acid found in such sour foods as pickled cucumbers, kombucha, kimchi, and yogurt, as well as for producing alcoholic beverages such as wine and beer. Fermentation also occurs within the gastrointestinal tracts of all animals, including humans.

The word "ferment" is derived from the Latin verb fervere, which means to boil.it is all about in etymology.

Fermentation, chemical process by which molecules such as glucose are broken down anaerobically. More broadly, fermentation is the foaming that occurs during the manufacture of wine and beer, a process at least 10,000 years old. The frothing results from the evolution of carbon dioxide gas, though this was not recognized until the 17th century.

How Does Fermentation Work?

Microorganisms survive using carbohydrates (sugars, such as glucose) for energy and fuel.

Organic chemicals like adenosine triphosphate (ATP) deliver that energy to every part of a cell when needed.

Microbes generate ATP using respiration. Aerobic respiration, which requires oxygen, is the most efficient way to do that. Aerobic respiration begins with glycolysis, where glucose is converted into pyruvic acid. When there’s enough oxygen present, aerobic respiration occurs.

Fermentation is similar to anaerobic respiration—the kind that takes place when there isn’t enough oxygen present. However, fermentation leads to the production of different organic molecules like lactic acid, which also leads to ATP, unlike respiration, which uses pyruvic acid.

Depending upon environmental conditions, individual cells and microbes have the ability to switch between the two different modes of energy production.

Organisms commonly obtain energy anaerobically through fermentation, but some systems use sulfate as the final electron acceptor in the electron transport chain.

Fermentation is all down to the actions of tiny natural microbes, who colonize and cultivate everything from our digestive systems, to this colorful spring in Yellowstone seen in the picture above, to the food and drink we eat. 

Microbes use carbohydrates (sugars, such as glucose) for energy to fuel their survival. To make use of that energy, organic chemicals like adenosine triphosphate (ATP) deliver it when needed to every part of a cell.

Microbes - and our own body cells - use respiration to generate ATP. The most efficient way for them to do that is through a process known as aerobic respiration, which requires oxygen.

Aerobic respiration starts with glycolysis, where glucose is converted into pyruvic acid. Then, when there's enough oxygen around, aerobic respiration takes place. 

Fermentation is similar to the kind of respiration that takes place when there isn't enough oxygen present, namely anaerobic respiration. However unlike respiration, which uses pyruvic acid, fermentation leads to the production of different organic molecules like lactic acid, which also leads to ATP.  

What are three types of fermentation? 

Lactic acid fermentation

Lactic acid fermentation is a metabolic process by which glucose and other six-carbon sugars (also, disaccharides of six-carbon sugars, e.g. sucrose or lactose) are converted into cellular energy and the metabolite lactate, which is lactic acid in solution.

Yeast strains and bacteria convert starches or sugars into lactic acid, requiring no heat in preparation. These anaerobic chemical reactions, pyruvic acid uses nicotinamide adenine dinucleotide + hydrogen (NADH) to form lactic acid and NAD+. (Lactic acid fermentation also occurs in human muscle cells. During strenuous activity, muscles can expend adenosine triphosphate (ATP) faster than oxygen can be supplied to muscle cells, resulting in lactic acid buildup and sore muscles. In this scenario, glycolysis, which breaks down a glucose molecule into two pyruvate molecules and doesn’t use oxygen, produces ATP.) Lactic acid bacteria are vital to producing and preserving inexpensive, wholesome foods, which is especially important in feeding impoverished populations. This method makes sauerkraut, pickles, kimchi, yogurt, and sourdough bread.

Ethanol fermentation/alcohol fermentation

Yeasts break pyruvate molecules—the output of the metabolism of glucose (C6H12O6) known as glycolysis—in starches or sugars down into alcohol and carbon dioxide molecules. Alcoholic fermentation produces wine and beer.

Ethanol fermentation, also called alcoholic fermentation, is a biological process which converts sugars such as glucose, fructose, and sucrose into cellular energy, producing ethanol and carbon dioxide as by-products.

Acetic acid fermentation

Starches and sugars from grains and fruit ferment into sour tasting vinegar and condiments. Examples include apple cider vinegar, wine vinegar, and kombucha

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.

Short notes on - Laminar Air Flow

 Short notes on - Laminar Air Flow

What is laminar air flow unit?

Laminar airflow is defined as air moving at the same speed and in the same direction,  By contrast, turbulent flow creates swirls and eddies that deposit particles on surfaces randomly and unpredictably.

                   A laminar flow cabinet or tissue culture hood is a carefully enclosed bench designed to prevent contamination of semiconductor wafers, biological samples, or any particle sensitive materials. 

                    Laminar airflow is defined as air moving at the same speed and in the same direction, with no or minimal cross-over of air streams (or “lamina”). By contrast, turbulent flow creates swirls and eddies that deposit particles on surfaces randomly and unpredictably.

Who invented Laminar Air flow?

Willis Whitfield

When Willis Whitfield invented the modern-day cleanroom 50 years ago, researchers and industrialists did not believe it at first. But within a few short years, US$50bn worth of laminar-flow cleanrooms were being built worldwide and the invention is used in hospitals, laboratories and manufacturing plants today.

What is the use of laminar air flow?

The process of laminar air flow can be described as airflow where an entire body of air flows with steady,in the laboratory, Laminar Flow Cabinets are commonly used for specialised work.Laminar airflow is used to separate volumes of air, or prevent airborne contaminants from entering an area. Laminar flow hoods are used to exclude contaminants from sensitive processes in science, electronics and medicine.

                      

                       

What is the principle of laminar flow?

laminar flow towards the user. Due to the direction of air flow, Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the users.

How do you get laminar air flow?

In a vertical clean bench,laminar air is then projected vertically over the work area's..

 In these instances, HEPA-filtered air mixes with and dilutes interior airborne contaminants inside the glove box. 

What is the function of laminar air flow?

The process of laminar air flow can be described as airflow where an entire body of air flows with steady, uniform velocity. Laminar Flow Cabinets work by the use of in-flow laminar air drawn through one or more HEPA filters, designed to create a particle-free working environment and provide product protection.

What is laminar air flow in microbiology?

Laminar Hood sometimes also known as Laminar Air Flow is an enclosed bench designed to prevent contaminations .

Laminar Air Flow are equipped with a UV lamp that should be turned on about 10-20 minutes before being used to sterilize the shell or cabinet or the surface of the Laminar Air Flow to avoid any kind of contaminations.

What is HEPA filter in laminar air flow?

In a laminar flow hood the air is passed through a HEPA (High Efficiency Particulates Air) filter which removes all airborne contamination to maintain sterile conditions

Procedure of Laminar air flow.?

Turn on the switch of UV light; leave the UV on for at least 30 minutes.

Turn OFF the UV light.

Turn ON the switch of visible light.

Turn ON the switch of Air Flow.

What is laminar flow in OT?

This is re-circulated under positive pressure into the operating theatre with surgically generated contaminants being continuously removed .

True laminar flow is only achieved when approximately 100% HEPA filter coverage occurs. 

in theatre more than 300 times per hour compared to standard positive pressure theatre rates of 15-25 air changes per hour. 

How does a laminar flow work? 

In fluid dynamics, laminar flow is characterized by fluid particles following smooth paths in layers.

There are no cross-currents perpendicular to the direction of flow, nor eddies or swirls of fluids.

Laminar flow is a flow regime characterized by high momentum diffusion and low momentum convection.

Laminar flow barriers :

Laminar flow hoods are used to exclude contaminants from sensitive processes in science, electronics and medicine.

Laminar flow cabinet :

Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the user.

A laminar flow cabinet or tissue culture hoodis a carefully enclosed bench designed to prevent contamination of semiconductor wafers,

Cleaning procedure of laminar air flow:

Cleaning a Vertical Laminar Flow Hood

Begin by taking the wipe and spraying disinfectant onto the wipe. 

After Cleaning :

Some laminar airflow hoods may have UV-C Germicidal Lamps for sterilization. 

Always remember to wash your hands afterward.

When cleaning is complete, dispose of wipes, gloves, and gown in the biohazard waste.

Why is laminar flow important?

It is the smooth flow of a fluid over a surface. Though a boundary layer of air "sticks" to a wing, the air overtop should be moving quickly and smoothly to reduce friction drag. Engineers want to design aircraft with laminar flow over their wings to make them more aerodynamic and efficient.

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DNA isolation protocol from Bacteria

 Short note on DNA isolation protocol from Bacteria concept by the biotechnify 

Abstract

This protocol uses phenol/chloroform method to purify genomic DNA without using commercial kits.

What is genomic DNA in bacteria?

Most bacteria have a genome that consists of a single DNA molecule (i.e., one chromosome) that is several million base pairs in size and is "circular" (doesn't have ends like chromosomes of eukaryotic organisms).

What is the principle of DNA extraction?

The DNA extraction process frees DNA from the cell and then separates it from cellular fluid and proteins so you are left with pure DNA. The three basic steps of DNA extraction are 1) lysis, 2) precipitation, and 3) purification.

Materials and Reagents

1. Tris base (Calbiochem-Behring)
   
2. Proteinase K (Sigma-Aldrich)
   
3. Phenol\chloroform (1: 1) (EM Science)
   
4. 200 proof ethanol (Pharmco-AAPER)
   
5. RNAase (Life Technologies, Invitrogen™)
   
6. Ethanol
   
7. SDS
   
8. EDTA
   
9. Tryptone
10. Yeast extract
11. NaCl
    
12. LB medium 
    
13. TE buffer 

Equipment

1. Tabletop centrifuge (Eppendorf)
   
2. 1.5 ml Eppendorf tube
   
3. Incubator
   
4. Gloves

ADDITIONAL Equipment :

• Waterbath or beaker and thermometer

• Mini Centrifuge

• Agarose Gel Equipment (optional)

• DNA Loading Buffer (optional)

• 70% Ethanol (Optional)

Procedure

1. Transfer 1.5 ml of the overnight E. coli culture (grown in LB medium) to a 1.5 ml Eppendorf tube and centrifuge at max speed for 1min to pellet the cells.
   
2. Discard the supernatant.
   Note: Remove as much of the supernatant as you can without disturbing the cell pellet.
   
3. Resuspend the cell pellet in 600 μl lysis buffer and vortex to completely resuspend cell pellet.
   
4. Incubate 1 h at 37 °C.
   
5. Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely mixed.
   Note: Do not vertex the tube—it can shear the DNA.
   
6. CAUTION: Phenol is a very strong acid that causes severe burns. Chloroform is a carcinogen. Wear gloves, goggles and lab coat, and keep tubes capped tightly. To be safe, work in the hood if possible.
   
7. Spin at max speed for 5 min at RT (all spins are performed at RT, unless indicated otherwise). There is a white layer (protein layer) in the aqueous: phenol/chloroform interface.
   
8. Carefully transfer the upper aqueous phase to a new tube by using 1 ml pipetman (to avoid sucking the interface, use 1 ml tip with wider mouth-cut 1 ml tip-mouth about ~2 mm shorter).
   
9. Steps 4-6 can be repeated until the white protein layer disappears.
   
10. To remove phenol, add an equal volume of chloroform to the aqueous layer. Again, mix well by inverting the tube.
    
11. Spin at max speed for 5 min.
    
12. Remove aqueous layer to new tube.
    
13. To precipitate the DNA, add 2.5 or 3 volume of cold 200 proof ethanol (store ethanol at -20 °C freezer) and mix gently (DNA precipitation can be visible).
    Note: DNA precipitation may simply diffuse, which is normal. Keep the tube at -20 degree for at least 30 min (the longer the better) and then spin it down (see Steps 15-16). You should see DNA pellet. It looks transparency when it is wet and turns to white when it becomes dry.
    
14. Incubate the tube at -20 °C for 30 min or more.
    
15. Spin at max speed for 15 min at 4 °C.
    
16. Discard the supernatant and rinse the DNA pellet with 1 ml 70% ethanol (stored at RT).
    
17. Spin at max speed for 2 min. Carefully discard the supernatant and air-dry the DNA pellet (tilt the tube a little bit on paper towel). To be faster, dry the tube at 37 °C incubator.
    
18. Resuspend DNA in TE buffer.
    Note: Large amounts of RNA will be present in the DNA sample. So, for subsequent reactions, for example, to digest plasmid DNA, add 1-5 μl (1 mg ml-1) RNAase to the digestion solution to completely remove RNA. Or, add RNAase directly to lysis buffer with a final concentration of 1 mg ml-1.
19. Check isolated Gemonic DNA on an agarose gel.
    Note: we expect to see bands with smear patterns from high to low MW range, although most of DNA fragments are accumulated at high MW on the gel. So, if you see most of DNA fragments are small, very likely your DNA got degraded.
    

Precautions :

 Material finely ground in liquid nitrogen should be immediately transferred into

the extraction buffer and must not be allowed to ‘sweat’..

 In chloroform : isoamyl alcohol extraction, the aqueous phase should be

carefully removed and organic phase re-extracted to ensure full recovery of

DNA. If no separation is observed between the two phases, may be due to high

concentration of DNA and /or cell debris in aqueous phase, dilution with more

digestion buffer and re-extraction is the solution.

 Care should be taken to do the operations as gently as possible. Vortexing,

pipetting using fine tips etc. should be avoided to prevent the shearing of DNA.

 DNA should not be over dried as resuspesion in TE become difficult.

 All the glassware, plastic ware, pestles and mortars etc. should be

decontaminated properly. Care should be taken to prevent cross-contamination.

 Blank extraction controls are carried out along with normal extractions to check

for any contamination.

TIME Required :

• Day 1: 2 hours

📲End. DNA isolation protocol from Bacteria