Principle Microbiology

GFP-Photo reaction:

· The GFP absorps mainly at 396 nm, followed by loss of proton in excited state to form phenolate.

· The main emmision occurs at 508 nm. Small amount of GFP absorps at 475 nm and emits green light at 508 nm.

· The GFP as well as green fluoroscent gene can be modified. The gene can be modified by mutation.

· The fluoroscent may be partially or completely lost. The mutation gives some misfolding of protein that generally fails to produce fluoroscence, but sometimes also produce higher emission.

Green Fluoroscent Protein:

· The GFP was first discovered from the jellyfish Aequorea victoria. But it is also present in other organisms.

· The Aequorea victoria contains two luminescent proteins.

1) Aequorin

2) GFP, i.e. Green Fluoroscent Protein

· Aequorin, when interacts with Ca+2, it emits flashes of the blue light.

· The GFP aquires energy from Aequorin and emits green light.

Structure of GFP:

· The GFP from Aequorea victoria has an 11 stranded beta-barrel structure, with a alpha-helix running up the axis of the barrel.

· The chromophore is attached to alpha-helix in the center of barrel, few amino acids make the chromophore.

· The chromophore has Serine, Tyrosine and Glycine at the position 65; 66 and 67 respectively.

· 4-(p-hydroxybenzylidene)-imidazolidin-5-one attached to protein backbone through 1 and 2 position of the ring.

· Chromophore has hydrogen-bonding with amino acid residue and water molecules.

Vaccines are any preparation which are made up of either whole microorganisms, macromolecules of microorganisms, DNAs or Recombinant Vector which can induce immunity in the host animal when given to it.

Vaccines are produced in many ways, most traditional of which is the embryonated hen's egg technique. But nowadays the place of embryonated hen's egg is being taken by the use of animal cell lines. Animal cell lines have various advantages over the other traditional techniques. Moreover animal cell lines also overcome some of the traditional problems faced during older techniques. There are also some limitations of the use of animal cell lines.

The whole article on the Use of animal cell lines in vaccine production can be read here as a powerpoint presentation on Google documents.

http://docs.google.com/leaf?id=0B0c57-C3kwyuOTdkZGVmZjMtZDA2YS00YjU4LTk2ZmYtZDY1NDg0YjU0ZTkz&hl=en

It contains following points...

What is a Vaccine?
Historical notes
Types of vaccines
Why cell lines?
Potential advantages
Ideal characteristics of cell lines used in vaccine production
Considerations Related to Specific Cell Lines
Production steps

I hope this article and the powerpoint helps you in one or another way...

- TRUNAL YATINDRA GUPTE

Recently one of my friend Nilesh Vaghela prepared a seminar on the topic Acid rain. I've uploaded his powerpoint presentation on my blog. I hope it'll help you people in one or other way. The seminar has covered following topics about Acid rain.
- Introduction to presenter and guide
- Definition
- Acid deposition
- Formation of Acid rain
- Measurement of Acid rain
- Effects of Acid rain
- Control measure of Acid rain
- References
- Thank you

The link to the powerpoint present on web is given below...

http://docs.google.com/present/view?id=dg4jc3qb_948jb7ng6f

The magnetic stirrer is used in many biological labs, including microbiology labs also. A magnetic stirrer is a laboratory device consisting of either a rotating magnet or stationary electromagnets creating a rotating magnetic field. This device is used to cause a stir bar immersed in a liquid to spin very quickly, agitating or mixing the liquid. A magnetic stirrer often includes a provision for heating the liquid. Stirrers are often used in laboratories, especially in the field of biology and microbiology. They are preferred over gear-driven motorized stirrers because they are quieter, more efficient, and have no moving external parts to break or wear out (other than the simple bar magnet itself).

Due to its small size, a stirring bar is more easily cleaned and sterilized than other stirring devices. Magnetic stirrers avoid two major problems with motorized stirrers. Firstly, motorized stirrers use lubricants, which can contaminate the reaction vessel and the product. Secondly, in motorized stirrers, the sealing of the connection between the rotating shaft of the stirrer and the vessel can be problematic, especially if a closed system is needed.

Magnetic stirrers also have drawbacks. For example, the limited size of the stirring bar means it can only be used for relatively small (under 4 liters) experiments. In addition, viscous liquids or thick suspensions are extremely difficult to mix using this method, although there are some stirrers with special magnets to overcome this problem.

See the following pictures on the top of the post:
<1> The magnetic stirrer
<2> The bar magnets used in stirrer

PCR principles and procedure

PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.

A basic PCR set up requires several components and reagents.

These components include:

* DNA template that contains the DNA region (target) to be amplified.

* Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target.

* Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.

* Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.

* Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.

* Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis.

* Monovalent cation potassium ions.


The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

Mary Mallon (September 23, 1869 – November 11, 1938), also known as Typhoid Mary, was the first person in the United States to be identified as a healthy carrier of typhoid fever. Over the course of her career as a cook, she is known to have infected 53 people, three of whom died from the disease. Her notoriety is in part due to her vehement denial of her own role in spreading the disease, together with her refusal to cease working as a cook. She was forcibly quarantined twice by public health authorities and died in quarantine. It is possible that she was born with the disease, as her mother had typhoid fever during her pregnancy.

Mallon was born in 1869 in County Tyrone, Northern Ireland, and emigrated to the United States in 1884. She worked as a cook in the New York City area between 1900 and 1907. She had been working in a house in Mamaroneck, New York for less than two weeks when the residents came down with typhoid. She moved to Manhattan in 1901 and members of the family for whom she worked developed fevers and diarrhea and the laundress died. She then went to work for a lawyer until seven of the eight household members developed typhoid. Mary spent months helping to care for the people she made sick, but her care further spread the disease through the household. In 1906, she took a position in Long Island. Within two weeks, six out of eleven family members were hospitalized with typhoid. She changed employment again and three more households were infected.

People catch typhoid fever after ingesting food or water which has been contaminated during handling by a human carrier. The human carrier is usually a healthy person who has survived a previous episode of typhoid fever but in whom the typhoid bacteria have been able to survive without causing further symptoms. Carriers continue to excrete the bacteria in their feces and urine. It takes vigorous scrubbing and thorough disinfection with soap and hot water to remove the bacteria from the hands. When typhoid researcher George Soper approached Mallon with the news she was possibly spreading typhoid, she adamantly rejected his request for urine and stool samples to ascertain whether she was a typhoid carrier. Soper left and later published his findings in the June 15, 1906 issue of the Journal of the American Medical Association.[1] On his next contact with her, he brought a doctor with him, but was again turned away. Mallon's denials that she was a carrier were based in part on the diagnosis of a reputable chemist who had found she was not harboring the bacteria. It is possible she was in temporary remission when tested. Moreover, when Soper first told her she was a carrier, the concept that a person could spread disease and remain healthy was not well known. During a later encounter in the hospital, he told Mary he would write a book about her and give her all the royalties; she angrily rejected his proposal and locked herself in the lavatory until he left.

Swine influenza (also called swine flu, hog flu and pig flu) refers to influenza caused by those strains of influenza virus, called swine influenza virus (SIV), that usually infect (is endemic in) pigs. As of 2009 these strains are all found in Influenza C virus and the subtypes of Influenza A virus known as H1N1, H1N2, H3N1, H3N2, and H2N3. Swine influenza is common in pigs in the midwestern United States (and occasionally in other states), Mexico, Canada, South America, Europe (including the United Kingdom, Sweden, and Italy), Kenya, Mainland China, Taiwan, Japan and other parts of eastern Asia. Transmission of swine influenza virus from pigs to humans is not common and does not always cause human influenza, often only resulting in the production of antibodies in the blood. The meat of the animal poses no risk of transmitting the virus when properly cooked. If transmission does cause human influenza, it is called zoonotic swine flu. People who work with pigs, especially people with intense exposures, are at increased risk of catching swine flu. In the mid-20th century, identification of influenza subtypes became possible, this allows accurate diagnosis of transmission to humans. Since then, fifty confirmed transmissions have been recorded, Rarely, these strains of swine flu can pass from human to human. In humans, the symptoms of swine flu are similar to those of influenza and of influenza-like illness in general, namely chills, fever, sore throat, muscle pains, severe headache, coughing, weakness and general discomfort. The 2009 flu outbreak in humans, known as "swine flu", is due to a new strain of influenza A virus subtype H1N1 that contains genes closely related to swine influenza. The origin of this new strain is unknown. However, the World Organization for Animal Health (OIE) reports that this strain has not been isolated in pigs. This strain can be transmitted from human to human, and causes the normal symptoms of influenza. Pigs can become infected with human influenza, and this appears to have happened during the 1918 flu pandemic and the 2009 flu outbreak.

The main raw materials used in beer production are as follows...


Malt
Malt adjuncts
Hops
Water

Malt :- The malt is prepared from carefully selected barley. This barley is first cleaned and then steeped in water for period up to two days. The excess water is then drained and the soaked barley is further incubated for periods of approximately four to six days to allow formation of a short rootlet and acrospire. This germination step allows the formation of highly viscous α - amylase, β - amylase and proteolytic enzymes, as well as flavor and color components.

At the end of incubation, the temperature is just raised to stop the germintaion without harming any enzyme, although high temperatures can be employed to obtain dark colored stout and bock beer fermentation.

Hence the green malt produced is carefully dried and stored. The preparation of good malt is an exacting task and it requires careful selection of barley and close supervision of malting process.
So most of the beer producing companies do not produce their own malt. But they rely on other companies who specialize in this art.

The other raw materials needed in beer production well be described soon.

The differential count gives us information on the number of different types of leukocytes in per 100 leukocytes.

The smear is made on the slide. First of all a drop is put on a slide. Then with the help of another slide the smear is prepared. The cells are counted one by one. No special slide like counting chamber or dilution fluids.

The cells are differentiated on the basis of nuclear lobes they contain. And the staining capacities of granules present in the cytoplasm.

The total count of WBCs or the leukocytes is required in the diagnosis of many diseases. The number of white blood cells is much higher in the blood. So it is requires to be diluted and the Turk's solution is used for the purpose.

Turk's solution contains glacial acetic acid which lysis the red blood cells which may interfere in the counting. And it also contains the Methylene blue which stains the nucleus of the white blood cells.

The counting is done in a specialized slides called Counting chambers. The number counter per volume is then multiplied with the dilution factor to get the final number of cells per ml in undiluted blood.

The counting chamber is shown in above figure.
Counting is done under high power microscope.

Links to the full notes for Transfer of oxygen to fermentation medium
and Raw materials for the fermentation medium



http://www.merifiles.com/uploads/transfer_of_oxygen_to_the_fermentation_medium_and_factors_affecting_it.jpg

&

http://www.merifiles.com/uploads/raw_materials_for_the_fermentation_medium.jpg

To change them from .jpg to .doc
- Go to the command prompt.
Start menu
Run
Command
- Go to the path of document
- Rename transfer_of_oxygen_to_the_fermentation_medium_and_factors_affecting_it.jpg transfer.doc


OR

- Rename raw_materials_for_the_fermentation_medium.jpg raw.doc

All micro-organisms require water, sources of energy, carbon, nitrogen, mineral elements and possibly vitamins plus oxygen if aerobic. On a small scale it is relatively simple to devise a medium containing pure compounds, but the resulting medium, although supporting satisfactory growth may be unsuitable for use in a large scale process.

On a large scale one must normally use sources of nutrients to create a medium which will meet as many as possible of the following criteria:

It will produce the maximum yield of product or biomass per gram of substrate used.
It will produce the maximum concentration of product or biomass.
It will permit the maximum rate of product formation.
There will be the minimum yield of undesired products.
It will be of a consistent quality and be readily available throughout the year.
It will cause minimal problems during media making and sterilization.
It will cause minimal problems in other aspects of the production process particularly aeration and agitation, extraction, purification and waste treatment.

The use of cane molasses, beet molasses, cereal grains, starch, glucose, sucrose and lactose as carbon sources, and ammonium salts, urea, nitrates, corn steep liquor, Soya bean meal, slaughter-house waste and fermentation residues as nitrogen sources, have tended to meet most of the above criteria for production media because they are cheap substrates. However, other more expensive pure substrates may be chosen.

It must be remembered that the medium selected will affect the design of fermenter to be used. For example, the decision to use methanol and ammonia in the single cell protein process developed by ICI plc necessitated the design of a novel fermenter design. The microbial oxidation of hydrocarbons is a highly aerobic and exothermic process. Thus, the production fermenter had to have a very high oxygen transfer capacity coupled with excellent cooling facilities. ICI plc solved these problems by developing an air lift fermenter. Equally, if a fermenter is already available this will obviously influence the composition of the medium.

A medium with a high viscosity will also need a higher power input for effective stirring. Besides meeting requirements for growth and product formation, the medium may also influence pH variation, foam formation, the oxidation-reduction potential, and the morphological form of the organism.

Historically, undefined complex natural materials have been used in fermentation processes because they are much cheaper than pure substrates. However, there is often considerable, batch variation because of variable concentrations of TR component parts and impurities in natural materials which cause unpredictable biomass and/or product yields. As a consequence of these variations in composition small yield improvements are difficult to detect. Undefined media often make product recovery and effluent treatment more problematical because not all the components of a complex nutrient source will be consumed by the organism. The residual components may interfere with recovery (chapter 10>and contribute to the BOD of the effluent.

Although manufacturers have been reluctant to use fined media components because they are more expensive, pure substrates give more predictable yields from batch to batch and recovery, purification and effluent treatment are much simpler and therefore cheaper. Process improvements are also easier to detect when pure substrates are used.

There are many industries which produce their products with the help of microorganisms. Some times the microorganisms themselves are the products. Microbes are used as they can carry out some processes which can't be carried out without the enzymes secreted by them.

Major products of industrial microbiology are as follows...

- Antibiotics
- Amino acids
- Organic acids
- Biopolymers
- Biosurfactants

Microbes as products...

- Nanotechnology
- Biosensors
- Biopesticides

The cheese production, beer production is not possible without micro organisms.

For a chemical compound To be an ideal chemotherapeutic agent used for treating microbial infections, it should have the following qualities:

(1)SELECTIVE TOXICITY :- The drug should demonstrate selective toxicity. This means that, at the optimum concentration, the drug should be toxic for the microorganism, but not for the host.

(2)ANTIMICROBIAL SPECTRUM :- The drug should be able to destroy or inhibit many kinds of pathocenic microorganisms. The larger the number of different microbial pathogenic soedes affected, the better.

(3)NO SIDE EFFECTS :- The drug should noç produce undesirable side effects, such as allergic reactions, nerve damage, irt of the kidney or damaging blood cells etc.

(4)NO KILLING EFFECT ON NORMAL FLORA :- The drug should not eliminate the normal icrobiat flora that inhabits the intestinal tract or other areas of the body. The normal flora also play an important role in preventing pathogens form growing.

(5)NO INACTiVATION :- If the drug is given orally, it should not be inactivated by stomach acids, and it should be absorbed into The body from the intestinal tract. If it is administrated by injection it shoud be inactivated by binding to blood proteins.

(6)NO DEVELOPMENT OF DRUG REStSTANCE :- The drug should inhibit microorganisms in such a way as to prevent the development of drug—resistant forms of pathogens.

INTRODUCTION
In modern usage, An antibiotic is a chemotherapeutic agent with activity against microorganisms such as bacteria, fungi or protozoa. The term "antibiotic" was coined by Selman Waksman in 1942 to describe any substance produced by a micro-organism that is antagonistic to the growth of other micro-organisms in high dilution. This original definition excluded naturally occurring substances, such as gastric juice and hydrogen peroxide (they kill micro-organisms but are not produced by micro-organisms), and also excluded synthetic compounds such as the sulfonamides (which are antimicrobial agents). Many antibiotics are relatively small molecules with a molecular weight less than 2000 Da. With advances in medicinal chemistry, most antibiotics are now modified chemically from original compounds found in nature, as is the case with beta-lactams (which include the penicillins, produced by fungi in the genus Penicillium, the cephalosporins, and the carbapenems). Some antibiotics are still produced and isolated from living organisms, such as the aminoglycosides; in addition, many more have been created through purely synthetic means, such as the quinolones.

OVERVIEW

Unlike previous treatments for infections, which often consisted of administering chemical compounds such as strychnine and arsenic, with high toxicity also against mammals, antibiotics from microbes had no or few side effects[citation needed] and high effective target activity. Most anti-bacterial antibiotics do not have activity against viruses, fungi, or other microbes. Anti-bacterial antibiotics can be categorized based on their target specificity: "narrow-spectrum" antibiotics target particular types of bacteria, such as Gram-negative or Gram-positive bacteria, while broad-spectrum antibiotics affect a wide range of bacteria.
The environment of individual antibiotics varies with the location of the infection, the ability of the antibiotic to reach the site of infection, and the ability of the microbe to inactivate or excrete the antibiotic. Some anti-bacterial antibiotics destroy bacteria (bactericidal), whereas others prevent bacteria from multiplying (bacteriostatic).
Oral antibiotics are simply ingested, while intravenous antibiotics are used in more serious cases, such as deep-seated systemic infections. Antibiotics may also sometimes be administered topically, as with eye drops or ointments.
In the last few years three new classes of antibiotics have been brought into clinical use. This follows a 40-year hiatus in discovering new classes of antibiotic compounds. These new antibiotics are of the following three classes: cyclic lipopeptides (daptomycin), glycylcyclines (tigecycline), and oxazolidinones (linezolid). Tigecycline is a broad-spectrum antibiotic, while the two others are used for Gram-positive infections. These developments show promise as a means to counteract the growing bacterial resistance to existing antibiotics.
Although potent antibiotic compounds for treatment of human diseases caused by bacteria (such as tuberculosis, bubonic plague, or leprosy) were not isolated and identified until the twentieth century, the first known use of antibiotics was by the ancient Chinese over 2,500 years ago. Many other ancient cultures, including the ancient Egyptians, ancient Greeks and medieval Arabs already used molds and plants to treat infections, owing to the production of antibiotic substances by these organisms, a phenomenon known as antibiosis.
Quinine became widely used as a therapeutic agent in the 17th century for the treatment of malaria, the disease caused by Plasmodium falciparum, a protozoanparasite.
Antibiosis was first described in 1877 in bacteria when Louis Pasteur and Robert Koch observed that an airborne bacillus could inhibit the growth of Bacillus anthracis. to the discovery of penicillin,The antibiotic properties of Penicillium sp. were first described in england by John Tyndall in 1875.However, his work went by without much notice from the scientific community until Alexander Fleming's discovery of Penicillin.
Modern research on antibiotic therapy began in Germany with the development of the narrow-spectrum antibiotic Salvarsan by Paul Ehrlich in 1909, for the first time allowing an efficient treatment of the then-widespread problem of Syphilis. The drug, which was also effective against other spirochaeta infections, is no longer in use in modern medicine.
Antibiotics were further developed in Britain following the discovery of Penicillin in 1928 by Alexander Fleming. More than ten years later, Ernst Chain and Howard Florey, Baron Florey|Howard Florey became interested in his work, and came up with the purified form of penicillin. The three shared the 1945 Nobel Prize in Medicine. In 1939, Rene Dubos isolated gramicidin, one of the first commercially manufactured antibiotics in use during World War II to prove highly effective in treating wounds and ulcers.
Prontosil, the first commercially available antibacterial antibiotic was developed by a research team led by Gerhard Domagk (who received the 1939 Nobel Prize in Physiology or Medicine for his efforts at the Bayer Laboratories of the IG Farben conglomerate in Germany. Prontosil had a relatively broad effect against Gram-positive Coccus but not against Enterobacteriaceae. The discovery and development of this first Sulfonamide drug opened the era of antibiotics.

Gel electrophoresis is a technique, used by microbiologist - biochemists - bio technologists, for the separation and analysis of various biochemical substances.

Here I've got a photograph of and gel electrophoresis unit from my college (Of course with permission of my teacher).

You can see the power supply wires (Black and Red) which gives electric current to the gel.
The gel can be seen in photo.

The separation of deoxyrebonucleic acid was going on while taking the picture (As per I know).
We can see two different bands of separated DNA in sky blue and nevy blue colours.

If you want notes on Gel electrophoresis or any technique related to microbiology please comment in any of my posts. I'll try to manage for you. Please dont forget to leave ur e-mail ID.

Cross-flow filtration (tangential filtration)

In the filtration processes previously described, the flow of broth was perpendicular to the filtration membrane.
Consequently, blockage of the membrane led to lower rates of productivity and/or the need for filter aids to be added, and these were serious disadvantages.
In contrast, an alternative which is rapidly gaining prominence both in the processing of whole fermentation broths and cell lysates is cross-flow filtration.
Here, the flow of medium to be filtered is tangential to the membrane, and no filter cake builds up on the membrane.

The benefits of cross-flow filtration are:

(a) Efficient separation : 99.9% cell retention.
(b) Closed system : For the containment of organisms with no aerosol formation.
(c) Separation is independent of cell and media densities, in contrast to centrifugation.
(d) No addition of filter aid.

(1) String discharge :- Fungal mycelia produce a fibrous filter cake which can easily be separated from the drum by string discharge.
Long lengths of string 1.5 cm apart are threaded over the drum and round two rollers.
The cake is lifted free from the upper part of the drum when the vacuum pressure is released and carried to the small rollers where it falls free.

(2) Scraper dircharge :- Yeast cells can be collected on a filter drum with a knife blade for scraper disc.
The filter cake which builds up on the drum is removed by an accurately positioned knife blade.
Because the knife is close to the drum, there may be gradual wearing of the filter cloth on the drum.

(3) Scraper discharge with precoating of the drum :- The filter cloth on the drum can be blocked by bacterial cells or mycelia of actinomycetes.
This problem is overcome by precoating the drum with a layer of filter-aid 2-10 cm thick.
The cake which builds up on the drum during operation is cut away by the knife blade.
Which mechanically advances towards the drum at a controlled slow rate.
Alternatively, the blade may be operated manually when there is an indication of ‘blinding’ which may be apparent from a reduction in the filtration rate.
In either case the cake is removed together with a very thin layer of precoat.

ROTARY VACUUM-FILTERS

Large rotary vacuum filters are commonly used by industries which produce large volumes of liquid which need continuous processing.
The filter consists of a rotating, hollow, segmented drum covered with a fabric or metal filter which is partially immersed in a trough containing the broth to be filtered.
The slurry is fed on to the outside of the revolving drum and vacuum pressure is applied internally so that the filtrate is drawn through the filter, into the drum and finally to a collecting vessel.
The interior of the drum is divided into a series of compartments, to which the vacuum pressure is normally applied for most of each revolution as the drum slowly revolves (~ 1 rpm).
How ever, just before discharge of the filter cake, air pressure may be applied internally to help ease the filter cake off the drum.
A number of spray jets may he carefully positioned so that water can be applied to rinse the cake. This washing is carefully controlled so that dilutions of the filtrate is minimal.
It should be noted that the driving force for filtration (pressure differential across the filter) is limited to one atmosphere (100 kN per meter square) and in practice it is significantly less than this.
In contrast, pressure filter can be operated at many atmospheres pressure. A number of rotary vacuum drum filters are manufactured.
Which differ in the mechanism of cake discharge from the drum.

(1)String discharge.
(2)Scraper discharge.
(3)Scraper discharge with precoating of the drum.