Principle Microbiology

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.

PRESSURE LEAF FILTERS

There are a number of intermittent batch filters usually called by their trade names. These filters incorporate a number of leaves, each consisting of a metal framework of grooved plates which is covered with a fine wire mesh, or occasionally a filter cloth and often precoated with a layer of cellulose fibres. The process slurry is fed into the filter which is operated under pressure or by suction with a vacuum pump. Because the filters are totally enclosed it is possible to sterilize them with steam. This type of filter is particularly suitable for ‘polishing’ large volumes of liquids with low solids content or small batch filtrations of valuable solids.

(i) Vertical metal-leaf filter

This filter consist of a number of verticel porous metal leaves mounted on a hollow shaft in a cylindrical pressure vessel. The solids from the slurry gradually build up on the surface of the leaves and the filtrate is removed from the plates via the horizontal hollow shaft. In some designs the hollow shaft can be slowly rotated during filtration. Solids are normally removed at the end of a cycle by blowing air through the shalt and into the filter leaves.

(ii) Horizontal metal—leaf filter
In this filter the metal leaves are mounted on a vertical hollow shaft within a pressure vessel. Often only the upper surfaces of the leaves are porous. Filtration is continued until the cake fills the spacc between the disc-shaped leaves or when the operational pressure has become excessive. At the end of a process cycle, the solid cake can be discharged by releasing the pressure and spinning the shaft with a drive motor.

(iii) Stacked-disc filter

One kind of filter of this type is the Metafilter. This is a very robust device and because there is no filter cloth and the bed is easly replaced, labour cost are low. It Consists of a number of precision-made rings which are stacked on a fluted rod. The rings are assembled on the rods.
The assembled stacks a placed in a pressure vessel which can be sterilized if necessary. The packs are normally coated with a thin layer of kieselguhr which is used as a filter aid. During use, the filtrate passes between the discs and is removed through the grooves of the fluted rods, while solids are deposited on the filter coating. Operation is continued until the resistance becomes too high and the solids are removed from the rings by applying back pressure via the fluted rods. Metafilters are primarily used for ‘polishing’ liquids such as beer.

Filters may be of two types.
(1) Batch Filters (2) Continuous Filters

Batch Filters

PLATE AND FRAME FILTERS
A plate and frame filter is a pressure filter in which the simplest form consists of plates and frames ar ranged alternately. The plates are covered with filter cloths or filter pads. The plates and frames are assembled on a horizontal framework and held together by means of a hand screw or hydraulic tam so that there is no leakage between the plates and frames which form a series of liquid-tight compartments. The slurry is fed to the filter frame through the continuous channel formed by the holes in the corners of the plates and frames. The filtrate passes through the filter cloth or pad, runs down grooves in the filter plates and is then discharged through outlet taps to a channel. Sometimes, if aseptic conditions are required, the outlets may lead directly into a pipe. The solids are retained within the frame and filtration is stopped when the frames are completely filled or when the flow of filtrate becomes uneconomicaly low.
On an industrial scale the plate and frame filter is one of the cheapest filters per unit of filtering space area requires the least floor space, but it is intermittent in operation (a batch process) and there may be considerable wear of filter cloths as a result of frequent dismantling.
This type of filter is most suitable for fermentation broths with a solids content and low resistance to filtration. It is widely used as a ‘polishing’ device in breweries to filter out residual yeast cells. following initial clarification by centrifugation or rotary vacuum filtration.
It may also be used for collecting high value solids that would not justify the use of a continuous filter. Because of high labour costs and the time involved in dismantling, cleaning and reassembly, these filters should not be used when removing large quantities of worthless solids from a broth.

It is common practice to use filter aids when filtering bacteria or other fine or gelatinous suspensions which prove slow to filter or partially block a filter. Kieselguhr (diatomaceous earth) is the most widely used material. It has a voidage of approxitnately 0.85, and when it is mixed with the initial cell suspension, improves the porosity of a resulting filter cake leading to a faster flow rate. Alternatively it may be used as an initial bridging agent in the wider pores of a filter to prevent or reduce blinding. The term ‘blinding’ means the wedging of particles which are not quite large enough to pass through the pores, so that an a fraction of the filter surface becomes inactive. The minimum quantity of filter aid to be used in filtration of a broth should be established experimentally. Kieselguhr is not cheap, and it will also absorb some of the filtrate, which will be lost when the filter cake is disposed.

The main methods of using the filter aid are:

1. A thin layer of kiesetguhr is applied to the filter to form a precoat prior to broth filtration.

2. The appropriate quantity of filter aid is mixed with the harvested broth. Filtration is started, to build up a satisfactory filter bed. The initial raffinate is returned to the remaining broth prior to starting the true filteration.

3. When vacuum drum filters are to be used which are fitted with advancing knife blades, a thick precoat filter is initially built up on the drum.

In some processes such as microbial biomass production, filter aids cannot be used and cell pretreatment by flocculation or heating must be considered. In addition it is not normally practical to use filter aids when the product is intracellular and its removal would present a further stage of purification.

The simple filtration apparatus is illustrated which consists of a support covered with a porous filter cloth. A filter cake gradually builds up as filtrate passes through the filter cloth. As the filter cake increases in thickness the resistance to flow will gradually increase. Thus, if the pressure applied to the surface of slurry is kept constant the rate of flow will gradually diminish. Alternatively, if the flow rate is to be kept constant the pressure will gradually have to be increased. The flow rate may also be reduced by blocking of holes in the filter cloth and closure of voids between particles, if the particles are soft and compressible. When particles are compressible it may not feasible to apply increased pressure.

Low temperature : -
The low temperature method is not used as the method of sterilization as it does not assure sterilization. Temperature below optimum depress the rate of metabolism and if the temperature is sufficiently low the growth and metabolism cease.
Low temperature can be used for preservation of culture.
Agar-slant culture of bacteria, yeasts and molds are stored for long period of time at 4 to 7 degree C.
Many bacteria and viruses are stored in deep-freeze unit at temperature of -20 to -70 degree C.
Many viruses and microorganisms, and stock of mammalian tissues are stored at -196 degree C in liquid nitrogen.

Hot air oven : -
This kind of dry heat sterilization is recomended when it is undesirable that steam make contact with the material to be sterilized. This is true for certain glasswares - Petriplates, Pipettes as well as for substances like oil, powder, etc. The apparatus employed is an electric/gas oven. Even the kitchen oven can be used. For laboratory glasswares 2 hours exposure to a temperature 160 degree C if enough for sterilization.

Incineration : -
Killing microorganisms by burning is one of the nice way to control them. It is the routine practice in microbiology lab when a transfer needle / loop is placed into the flame of the Bunsen burner.
A note of caution should be added here - When the transfer needle / loop is sterilized, care should be taken to prevent the droplets flying off from needle / loop, as they may contain viable microorganisms which may be pathogenic.
The danger can be avoided by using electric heat coil with a tube surrounding it into which transfer needle / loop can be inserted.
This method is used for carcasses, infected laboratory animals and other infected material to be disposed off. Care should be taken that exhaust fumes do not contain viable microorganisms into atmosphere.

Boiling water : -
Boiling water destroyes all the vegetative cells. But this method does not assure complete sterility of contaminated material or the object because the resistant bacterial spores can withstand this condition for many hours.


Pasteurization : -
Milk and some other beverages are subjected to a controlled heat treatment called pasteurization. In which even all vegetative cells are not killed. But only some of them which may be pathogenic are killed. The pasteurized milk in not the sterilized milk.

Fractional sterilization : -
Some mocrobiological materials can not be heated above 100 degree C. But if still they can withstand the temperature of free flowing steam (i.e. 100 degree C) they can be sterilized by fractional sterilization / Tyndalization. In this method the material is heated with steam of 100 degree C on three successive days with incubation period in between. The vegetative cell will die on the 1st exposure with heat and the spores will germinate in incubation period. This germinated spores can be killed on second exposure with heat. If the spore will germinate it will become vegetative cell which can be easily destroyed, but if it doed not germinate sterilization will not be obtained. After three exposures sterilization is obtained. For this method one can use steam arnold. Even an autoclave with free flowing steam can be used for this purpose.

Steam under pressure : -

High temperature can be used in two ways.
1) Moist heat
2) Dry heat

Moist heat has four subtypes
- Steam under pressure,
- Fractional sterilization/Tyldallization,
- Boiling water,
- Pastuerization.

Dry heat has two subtypes
- Hot air oven,
- Incineration.

Control of microorganism is a very important process.
Microorganisms can be controlled by physical as well as chemical methods.
Physical methods are Temperature, Radiation, Dessication, Surface tension and interfacial tension, etc.
Chemical method implies various chemicals to destroy microorganisms (MO now on.)

Bacterial Growth curve

Bacteria, when transferred from one medium to another, shows characterstic growth curve.
It shows four distinct phase of growth.
Lag phase
Log or exponential phase
Stationary phase
Decline or death phase

Lag phase :- When the bacteria are transferred from one medium to another they do not show sudden growth as they may be depleted of energy. The nutrients may be different in two media. Organisms synthesize new enzymes to utilize new nutrients. Organisms increase in size but show no change in number.
Log phase :- In this phase bacteria divide exponentially. The graph shows curve which means not all the bacteria multiply simultaneously. After some time period the no. of division fall down.
Stationary phase :- In this phase bacterial population does not change with time - No. of bacteria born are same as no. of bacteria died.
Decline phase :- In this phase bacteria die exponentially. Bacterial population fall down as inverse of log phase. This phase come due to accumulation of waste products and depletion of nutrients.

Bacteriological media contain nutritional requirements for bacteria.
It contains peptones, meat extract and yeast extract.
Agar is used as a solidifying agent and not as nutritive.
Medium may be liquid ( Nutrient broth medium) or solid (Nutrient agar medium) or even semisolid.
Special kind of media are also there like Selective media, Differential media, Maintenance media, Enumeration media, Characterization media, Assey media. etc.

Like the all other form of life, bacteria also have some nutritional requirements.
They need energy source - It may be Chemical compound,
It may be Radient energy (Light).
They need carbon source - It may be Organic compound,
It may be Carbon dioxide.
They need nitrogen source - It may be Atmospheric nitrogen,
It may be Inorganic nitrogen compounds - NItrates, Nitrites, ammonium salts,
It may be Organic nitrogen compounds - Amino acids.
They need electorn donor - It may be inorganic compound,
It may be organic compound.
They need Oxygen source - Water, Various nutrients or molecular oxygen.
They need phosphorus source - Phosphate, Nucleotides, Nucleic acids, Phospholipids, Teichoic acids and other compounds.
They need sulfur source - Inorganic/organic sulfur compounds or even elemental sulfur.
They also require metal ions for their normal growth and metabolism, Vitamins which may act as coenzymes for various enzymes.
And they also require WATER for the growth.
There are some physical requirements for microorganisms. Like temperature, pH, Gaseous requirements, ect.

Serial dilution method :-
The original culture suspension is diluted more one time - Serial dilution, in tubes or in appropriate medium. Because of the reduction in the no. of the bacteria due to dilution isolation is obtained. When greatly diluted the speciman contains only few organisms of only one species. The culture obtained is conserved by the spread plate or streak plate method.

Pour plate method :-
The speciman is serially diuted - More than one times as the density of the original speciman is unknown. The agar medium is maintained at the temperature of 45 C and the diluted suspension is inoculated and mixed well. The liquid state permits the through out distribution within the medium. The inoculated medium is poured in the sterile empty petriplate and allowed to solidify & then incubated. Next day the surface colonies as well as submerged colonies are observed.

Spread plate method :-
A serially diluted inoculum is transferred over the surface of the solid medium and then spreaded uniformly with a sterile bent glass spreader. But this technique does not ensure a nice isolation when dense suspension is used.