Principle Microbiology

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.

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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.