Microbial Control by Physical Methods

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Chapter: Pharmaceutical Microbiology : Microbial Control by Physical and Chemical Methods

The physical methods related to microbial control (or growth) are as enumerated under : (a) Heat, (b) Moist Heat, (c) Pasteurization, (d) Dry-Heat Sterilization, (e) Filtration, (f) Cold, (g) Desiccation, (h) Osmotic Pressure, and (i) Radiation.



The physical methods related to microbial control (or growth) are as enumerated under :


(a) Heat,

(b) Moist Heat,

(c) Pasteurization,

(d) Dry-Heat Sterilization,

(e) Filtration,

(f) Cold,

(g) Desiccation,

(h) Osmotic Pressure, and

(i) Radiation.


All these individual methods shall now be treated separately in the sections that follows :


(a). Heat


Heat represents probably the most common effective, and productive means whereby organisms are almost killed. In fact, it is a usual practice to have the laboratory media, laboratory glasswares, and hospital surgical instruments adequately sterilized by heat i.e., moist heat in an electric autoclave.


Salient Features. Following are the salient features of heat controlled microbes, namely :


(1) Most economical and easily controlable means of microbial growth.


(2) Usually kill microbes by causing denaturation of their respective enzymes.


(3) Heat resistance capacity of the organism must be studied carefully and taken into consideration.


(4) Thermal Death Time (TDT). TDT is referred to as the minimal length of time whereby all microbes present in a liquid culture medium will be killed at a given temperature.


(5) Thermal Death Point (TDP). TDP designates the lowest temperature at which all of the microorganisms present in a liquid suspension will be killed in just 10 minutes. In fact, heat resistance predominantly varies amongst the different range of organisms ; besides, these glaring differences may be duly expressed via the concept of thermal death point (TDP).


However, it is pertinent to state here that both TDP and TDT are equally vital, important, and useful guidelines which essentially indicate the actual prevailing severity of treatment needed to kill a given population of organisms.


(6) Decimal Reduction Time [DRT or D-Value]. DRT or D-Value represents a 3rd concept which is directly associated with the organism’s extent of heat resistance. In fact, it is very much equivalent to the time (minutes), whereby almost 90% of the population of prevailing microbes at an exact specified temperature shall be killed as illustrated in Fig. 7.1, having DRT of 1 minute. It is, however, pertinent to mention here that DRT is of an extreme importance and usefulness in the ‘canning industry’ dealing with fruit concentrates, fruit pulps, fruit slices, baked beans, corned-beef, fish products, fish chuncks, baby corns, lentils, and the like.

[Redrawn From : Tortora GJ et. al. : Microbiology : An introduction., The Benjamin/Cummings Pub. Co. Inc. New York, 5th edn., 1995].


The curve in Fig. 7.1 is plotted logarithmically (as shown by solid line), and arithmatically (as shown by broken line). In this particular instance, the microbial cells are found to be dying at a rate of 90% min–1.


b. Moist Heat


It is a common practice to make use of ‘heat’ in the process of sterilization either in the form of ‘moist heat’ or ‘dry heat’.


It has been duly proved and established that the so called ‘moist heat’ invariably kills microbes at the very first instance by the process known as ‘coagulation of proteins’, that is eventually caused by the specific cleavage of the H-bonds which critically retain the protein in its 3D-structure*. Interest-ingly, one may visualize the phenomenon of protein coagulation/denaturation rather more vividly in the presence of water.


‘Moist heat’ sterilization may be achieved effectively by the following widely accepted known methods, such as :


(a) Boiling,


(b) Autoclaving, and


(c) Pasteurization.


Each of the aforesaid method of moist-heat sterilization shall now be treated individually in the sections that follows :


(a) Boiling


Boiling at 100°C at 760 mm atmospheric pressure is found to kill particularly several varieties of vegetative states of microbial strains, a good number of viruses and fungi ; besides their ‘spores’ within a span of 10 minutes only. It is quite obvious that the ‘unpressurized’ i.e., free-flowing steam is practically equivalent to the prevalent temperature of boiling water (i.e., 100°C). It has been revealed that the endospores plus certain viruses are evidently not destroyed in such a short duration of 10 minutes.



(a) A typical hepatitis virus may even survive upto a duration of 30 minutes of continuous boiling at an atmospheric pressure.

(b) Likewise, there are certain microbial endospores that have been offered resistance to boiling for more than 30 hours.


Conclusion. Boiling for a couple of minutes will certainly kill organisms present in a Baby’s Feeding Bottle + Nipple, food products, drinking water relatively safer for human consumption.


(b) Autoclaving


The most reliable sterilization with moist heat prominently requires such ranges of temperature that are critically above the boiling water i.e., above 100°C. These high temperatures [120 ± 2°C] are most conveniently accomplished by moist steam under positive pressure usually in an ‘autoclave’. One may make use of ‘autoclaving’ as a means of sterilization unless the drug substance or material to be sterilized can suffer serious type of damage either by heat or by moisture. In fact, higher the pressure inside the autoclave, the higher will be the temperature inside the autoclave.


Examples. The following are two typical sets of examples viz.,


(a) Relationship between pressure and temperature of steam at sea level. It has been adequately proved that—‘the higher the pressure created inside the autoclave, the higher would be the attainable temperature inside the autoclave’.


When the free-flowing stream at a prevailing temperature of 100°C is subjected under pressure of 1 atmosphere above the sea-level pressure i.e., 15 pounds pressure per square inch (psi), the tempera-ture inside the autoclave happens to rise upto 121°C, which is an usual and common parameters em-ployed in the sterilization of food products and surgical instruments. One may also work at relatively lower/higher pressure (psi) vis-a-vis lower/higher temperatures (°C) as clearly given in Table : 7.1.


Table 7.1. Relationship Between Pressure and Temperature of Steam at Sea Level*

Figure 7.2 illustrates the beautiful elaborated diagramatic representation of an autoclave.

In a broader perspective, ‘sterilization’ in an autoclave is considered to be most effective par-ticularly in a situation when the microbes either contact the steam directly or are adequately contained in a small volume of aqueous (mostly water) liquid. Importantly, under such a critical experimental param-eters (i.e., steam at a pressure of 15 psi at 121°C) all the microbes would be killed while their endospores in almost within a span of 15 minutes.


Applications of an Autoclave. The various applications of an autoclave are as enumerated under :


(1) To sterilize culture media for the identification and propagation of pure strains of microor-ganisms and yeasts.


(2) To sterilize various surgical stainless steel instruments that are required for most of the sur-gical procedures, dental procedures, obstretrics etc.


(3) To sterilize various types of surgical dressings, gauzes, sutures etc.


(4) To sterilize a host of IV applicators, equipments, solutions, and syringes as well.


(5) To sterilize transfusion equipment(s) and a large number of other alied items that can con-veniently withstand high pressures and temperatures.


(6) When the ‘large industrial premises’ make use of the autoclaves, these are knwon as re-torts, whereas, the small domestic applications invariably employ pressure cookers (both based on exactly the same principles) for preparation of food* and canning of processed food products.


Important Aspects. In a situation, when we essentially look for extended heat requirement so as to specifically reach the exact centre of the solid materials viz., canned meats, fish (tuna), due to the fact that such materials fail miserably to develop the most desired efficient convection currents which invariably take place in the body of liquids.


Therefore, the particular heating of large containers/vessels does essentially require extra time period (in minutes) as given in Table 7.2.


Table 7.2. Overall Effect of Container Size upon Autoclave Sterilization Times (Minutes) for Liquid Solutions**.


(i) The autoclave sterilization times in the autoclave very much include the time required for the contents of the containers to perfectly reach the sterilization temperatures.


(ii) Obviously, for a very small container this is only 5 minutes or even less, whereas for a 9 L capacity fermentation bottle it might be as high as ~ 70 minutes.


(iii) All containers that are supposed to be sterilized by ‘autoclave’ are invariably filled only upto 3/4th the total volume i.e., their actual capacity.


Salient Features. The salient features of ‘autoclave sterilization’ are briefly stipulated as under :


(1) In order to sterilize duly the surface of a solid, one must allow the ‘steam’ to actually contact the same. Nevertheless, particular care must be taken to allow the perfect sterilization of bandages, dry-glasswares, and the like so as to ascertain that steam gets into contact with all the exposed surfaces.

Example. Aluminium foil does not allow the passage of steam to pass across (i.e., impervious), and hence must be avoided to wrap such materials meant to be sterilized ; instead, one may freely make use of brown wrapping paper (cellulose).


(2) Trapped Air. All necessary precautions and requisite care must be taken to get rid of any trapped air strategically located at the bottom of a ‘dry container’, due to the fact that the ‘trapped air’ shall not be replaced by ‘steam’ at any cost, which being lighter than air. However, one may just visual-ize imaginatively the so called ‘trapped air’ as a mini-hot air oven, that would eventually require not only a higher temperature but also a much longer duration to sterilize materials.


Based on the actual experience one may specifically tackle such containers which have a ten-dency to trap air must be positioned in a ‘tipped state’ in order that all the steam shall ultimately help to force out the air.


Note. Importantly, such products which obstruct penetration by moisture viz., petroleum jelly, mineral oil (furnace oil) are not usually sterilized by the same methods as adopted to sterilize aqueous solutions.


(c) Pasteurization


Pasteurization refers to ‘the process of heating of a fluid at a moderate temperature for a definite period of time to destroy undesirable microorganisms without changing to any extent the chemical composition.’


Example. In pasteurization of milk, pathogenic organisms are invariably destroyed by heating at 62° C for a duration of 30 minutes, or by ‘flash’ heating to higher temperatures for less than 1 minute, which is otherwise known as high-temperature short time (HTST) pasteurization.


In a broader perspective the pasteurization of milk, effectively lowers the total bacterial count of the milk by almost 97 to 99%, due to the fact that the most prevalent milk-borne pathogens viz., Tubercle bacillus*, and Samonella, Streptococcus, and Brucella organisms, fail to form ‘spores’, and are quite sensitive to heat.


It may, however, be observed that several relatively heat-resistant (thermoluric) microorganisms do survive pasteurization, and these may ultimately fail to :


·        Cause refrigerated milk to turn sour (spoil) in a short span of time, and


·        Cause any sort of disease in humans.


Ultra-High-Temperature (UHT) Treatments. Sterilization of milk is absolutely different from pasteurization. It may be duly accomplished by UHT treatments in order that it can be most easily and conveniently stored even without any sort of refrigeration. So as to maintain the first order ‘organoleptic characteristic features’** of fresh milk and to avoid attributing to the milk a prevalent cooked taste, the UHT system gained reasonable qualified success and hence due recognition across the globe, whereby the liquid milk never touches a surface hotter than the milk itself during the course of heating by steam.


Methodology. The various steps involved are as follows :


(1) Milk is allowed to fall in a thin-film vertically down through a stainless-steel (SS) chamber of ‘superheated steam’, and attains 140°C in less than 1 second.


(2) Resulting milk is adequately held for a duration of only 3 seconds duly in a ‘holding tube’.


(3) Ultimately, the pre-heated milk is cooled in a ‘vacuum chamber’, wherein the steam simply flashes off.


(4) The above stated process [(in (3)] distinctly enables the milk to raise its temperature from 74—140°C in just 5 seconds, and suddenly drops back to 74°C again.


Summararily, the very concept of equivalent treatments* clearly expatiates the particular rea-sons of the various methods of killing microbes, such as :


Pasteurization : At 63°C for 30 minutes ;


HTST-Treatment : At 72°C for 15 seconds ;


UHT-Treatment : At 140°C for < 1 second ;


(d) Dry-Heat Sterilization


It is a well known fact that microorgansims get killed by dry heat due to the oxidation effects.


Direct Flaming. Direct flaming designates one of the most simple method of dry-heat sterili-zation. In reality, the dry-heat sterilization is mostly used in a ‘microbiology laboratory’ for the steri-lization of the ‘inoculating loops’, which is duly accomplished by heating the loop wire to a ‘red-glow’, and this is 100% effective in actual practice. Likewise, the same principle is even extended to the process of ‘inceneration’ to sterilize as well as dispose of heavily contaminated paper bags, cups, and used dressings.


Hot-Air Sterilization. It may be regarded as another kind of dry-heat sterilization. In this particular process, the various items need to be sterilized are duly kept in an electric oven, preferably with a stainless-steel chamber inside, and duly maintained at 170°C for a duration of approximately 2 hours (to ensure complete sterilization).


It has been adequately observed that the longer the period plus higher temperature are needed profusely due to the fact that the heat in water is more rapidly passed onto a ‘cool body’ in comparison to the heat in air.


Example. The experience of exposing the ‘finger’ in a boiling water at 100°C (212°F) vis-a-vis exposing the same ‘finger’ in a hot-air oven at the same tempearture for the same duration.


(e) Filtration


Filtration may be defined as ‘the process of removing particles from a solution by allowing the liquid position to pass through a membrane or other particle barrier’. In reality, it essentially contains tiny spaces or holes which exclusively allow the liquid to pass but are too small to permit the passage of the small particles.


In other words, one may also explain ‘filtration’ as the process of a liquid or gaseous substance via a screen-like material having suitable pores small enough to retain the microorganisms (bacteria). A vacuum which is formed in the ‘receiver flask’ actually aids by means of gravity to suck the liquid via the filter medium engaged. However, in actual practice the phenomenon of filtration is invariably em-ployed to sterilize the specific heat sensitive substances, namely : culture media ; vaccines ; enzymes ; and several antibiotic solutions.


High-Efficiency Particulate Air (HEPA) Filters. HEPA-Filters are mostly used to get rid of practically all microbes that happen to be larger than 0.3 μm in diameter.


Examples. HEPA-Filters are largely used in :


(a) Intensive-Care Units [ICUs] in specialized hospitals treating severe Burn cases.


(b) In Sterile Zones of High-Value Antibiotic Preparations, Packaging, IV-injections, and other such sensitive sterile preparations.


Membrane Filters. In the recent past, technologically advanced membrane filters made up of either Cellulose Esters or Plastic Polymers have been employed profusely for the laboratory and industrial applications as shown in Fig. 7.3 and 7.4.


Explanation for Fig. 7.4 :


(1) The sample to be filtered is duly loaded into the ‘upper chamber’, and consequently forced through the strategically placed membrane filter.


(2) The pores present in the membrane filter are definitely much smaller in comparison to the microorganisms ; and, therefore, the microorganisms present are obviously retained upon the surface of the filter.


(3) Sterilized sample (free from microbes) may now be decanted conveniently from the ‘lower chamber’.


Specifications of Membrane Filters. Membrane filters usually have a thickness of 0.1 μm, and having almost uniform pores. However, in certain commercially available brands, the film is duly irradiated so as to generate extremely uniform holes, where the radiation particles have made its passage, are critically etched in the plastic. The pores of membrane filters usually range between 0.22 to 0.45 μm, intended for microorganisms.


Note. (1) Certain highly flexible microbes viz. spirochaetes, and the wall-less bacteria viz., mycoplasma, may sometimes pass through such membrane filters.


(2) To retain certain viruses and large-sized protein molecules are duly retained by such filters with pore size as small as 0.01 μm.


(f) Cold


It has been critically observed that the overall effect of ‘low temperature’ upon the microorgan-isms exclusively depends on the specific organism and the intensity of the application.


Example. At temperatures ranging between 0–7°C (i.e., the ordinary refrigerator), the actual rate of metabolism of majority of microorganisms gets reduced substantially to such an extent that they are rendered incapable of either synthesizing toxins* or causing reproduction.**


Thus, one may conclude that ‘ordinary refrigeration’ exerts a distinct bacteriostatic effect i.e., stops the multiplication vis-a-vis growth of microbes.


Psychotrophs***, however, are found to grow appreciably but slowly particularly at the refrigerator temperature conditions ; and may change the very appearance and taste of food products after a certain lapse of time.


Salient Features. The various salient features of microbes in a ‘cold’ environment are as follows :


(1) A few microbes may even grow at sub-freezing temperatures (i.e., below the freezing temperature).


(2) Sudden exposure to sub-freezing temperatures invariably render bacteria into the ‘dormant-state’; however, they do not kill them (bactericidal effect) ultimately.


(3) Gradual Freezing is observed to be quite harmful and detrimental to microorganisms, per-haps due to the fact that the ice-crystals which eventually form and grow do disrupt the cellu-lar as well as the molecular structure of the microorganisms.


(4) Life-Span of Frozen Vegetative Microbes—Usually remain active for a year upto 33% of the entire initial population, whereas other microbial species may afford relatively very scanty survival rates.


(g) Desiccation


In order to have both normal growth and adequate multiplication the microorganisms do re-quire water. Desiccation represents a typical state of microbes in the absence of water ; however, their growth and reproduction remain restricted but could sustain viability for several years. Interestingly, as soon as ‘water’ is duly made available to them the said organisms resume their usual growth and divi-sion as well. This highly specific ability has been adequately employed in the laboratory manipulations whereby the microbes are carefully preserved by lyophilization.*


It has been duly observed that the ensuing resistance of the vegetative cells to undergo the phenomenon of desiccation changes with the specific species as well as the microorganism’s environment.


Example : Gonorrhea** organism, Neisseria gonorrhoeae (Gonococcus), possess an ability to withstand dryness only upto a duration 60 minutes hardly ; whereas, Tuberculosis*** bacterium, Mycobacterium tuberculosis (Bacillus) may even remain completely viable for months together at a stretch.


Important Points : Following are certain important points which should always be borne in mind :

(a) An invariably susceptible microbe is found to be appreciably resistant when it gets duly embedded in pus cells, mucous secretions, and in faeces.

(b) In contract to microbes the viruses are usually found to be quite resistant to the phenomenon of ‘desiccation; however, they do not exhibit resistance comparable to the bacterial endospores.

(c) Importantly, in a typical hospital environment (setting) the presence and subsequent ability of some particular dried bacteria and endospores do remain absolutely viable, such as : beddings, clothings, dust particulate matters, and above all the disposable (used) dressings from patients may contain infectious organisms strategically located in dried pus, faecal matter, mucous secretions, and urine.


(h) Osmotic Pressure


Osmotic pressure refers to–‘the pressure which develops when two solutions of different concentrations are duly separated by a semipermeable membrane’.


In actual age-old practice, the preservation of food products viz., pickles, fruits, are duly accom-plished by the use of high-concentrations of salts and sugars which eventually exert their effects on account of the osmotic pressure. The most logical and probable underlying mechanism being the creation of an extremely hypertonic environment due to the presence of these substances (salts and sugars) at high concentrations that enables water to leave the microbial cell precisely. In fact, the preservation afforded by the osmotic pressure very much resembles to that caused by desiccation (see Section, besides, the glaring fact that both processes evidently deny the microbial cell of the requisite quantum of moisture essentially required for its normal growth. Dehydration of the microbial cell actu-ally renders the plasma membrane to shrink away from the respective cell-wall (i.e.,plasmolysis), whereby the consequent cell stops growth (and hence reproduction), and it may not cause an instant death. In a broader perspective, the fundamental principle of osmotic pressure is largely exploited in the prolonged preservation of food products.


Examples : (a) Concentrated Salt Solutions (Brine Solution) may be used profusely in the preservation and cure of meats, fish, vegetables, pickles etc.

(b) Concentrated Sugar Solutions (Sugar Syrup) may be employed, extensively in the preser-vation of lime juice, fruits etc.


(i) Radiation


Radiation refers to — ‘any form of radiant energy emission or divergence, as of energy in all directions from luminous bodies, radiographical tubes, particle accelerators, radioactive ele-ments, and fluorescent substances’.


It has been established beyond any reasonable doubt that radiation exerts its various effects on the cells, depending upon its wavelength, intensity, and duration as well. Generally, one may come across two kinds of radiation which would cause a bactericidal effects on microbes, or usually referred to as the ‘sterilizing radiation’, namely :


(a) Ionizing Radiation, and

(b) Nonionizing Radiation.


Each of the aforesaid types of radiation shall be treated individually in the sections that follows :


Ionizing Radiation


The ionizing radiation normally possess a wavelength distinctly shorter in comparison to the nonionizing radiation (size < 1 nm) e.g., γ-rays, X-rays, or high-energy electron beams.


Figure 7.5 vividly depicts that the said ionization radiation invariably carries a significant quan-tum of energy ranging between 10–5 nm (γ-rays) to 10–3 nm (X-rays).


γ-Rays : These are emitted by radioactive cobalt (Co),


X-Rays : These are produced by X-ray machines, and


Electron Beams : These are generated by accelerating electrons to high energies in special machines.


Visible light plus other forms of radiant energy invariably radiate via space as waves of various lengths.


Ionizing radiation viz., γ-rays and X-rays possess a wavelength shorter than 1 nm.


Nonionizing radiation viz., UV-light has a wavelength ranging between 1–380 nm, where the visible spectrum commences.


Salient Features. The various salient features of the Ionizing Radiation are as stated under :


(1) The γ-rays usually penetrate deeply but would essentially require reasonably longer dura-tion, extended to several hours, for the sterilization of relatively large masses.


(2) High-energy electron beams do possess appreciably lower penetrating power ; however, need only a few seconds of exposure to cause sterilization.


(3) Major causative effect of ionizing radiation being its distinct ability to the ionization of water, which in turn gives rise to highly reactive hydroxyl radicals [OH•]*. Interestingly, these radi-cals critically interact with the cellular organic components, especially the DNA, and thereby kill the cell ultimately.


(4) High-energy electron beams (ionizing radiation) has recently gained an enormous world-wide acceptance, recognition, and utilities for the exclusive sterilization of such substances as : pharmaceuticals, disposable dental materials, and disposable medical supplies. A few typical examples are : plastic syringes, catheters, surgical gloves, suturing materials.


Note. Radiation has virtually replaced ‘gases’ for the ultimate sterilization of these items.


Nonionizing Radiation


Predominantly the nonionizing radiation possesses a distinct wavelength much longer than that of the corresponding ionizing radiation, invariably greater than about 1 nm.


Example : UV-light : The most befitting example of the nonionizing radiation is the UV-light, which is able to cause permanent damage to the DNA of exposed cells by virtue of creation of newer additional bonds between the ‘adjacent thymines’ strategically present in the DNA-chains, as illus-trated in Figure : 7.6. The said figure evidently shows the formation of a thymine dimer after being exposed duly to the UV-light whereby the adjacent thymines may be rendered into a cross-linked entity. Importantly, in the absence of the visible light, this particular mechanism is usually employed by a cell to afford the repair of the prevailing damage caused.

In reality, these ‘thymine dimers’ are found to cause effective inhibition in correcting replica-tion of the DNA in the course of division (reproduction) of the cell. It has been duly established that the UV-wavelengths at nearly 260 nm are most effective and useful for killing microbes due to the fact that these are exhaustively absorbed by the cellular DNA.


Advantages of UV Light : are as given under :


(a) It controls and maintains the miroorganisms in the air.


(2) A ‘UV-Radiation Lamp’ or a ‘Germicidal Lamp’ is abundantly and profusely employed in a variety of such sensitive areas as : operation theaters, hospital rooms, nurseries, and cafeterias.


(3) UV Light or Radiation is invariably employed to sterilize a plethora of highly sensitive biological products commonly used in the therapeutic armamentarium, such as : serum, toxins, and a variety of vaccines.


(4) UV Light is also employed to sterilize the drinking water in homes, hospitals, and public places.


(5) UV Radiation is also used for the sterilization of the ultimate treated ‘municipal-waste waters’ for agriculture and horticulture purposes.


Disadvantages of UV Light : These are as stated under :


(1) UV Radiation is found to be not very penetrating in nature ; and, therefore, the microorgan-isms intended to be killed should be exposed almost directly to the UV-rays.


(2) Besides, such microbes that are adequately shielded (protected) by means of textiles, col-oured, glass, and paper (i.e., textured cellulose materials) are observed to be least affected by the UV radiation.


(3) Serious Problem. In fact, UV light poses a serious problem in causing permanent damage to human eyes on direct exposure, besides, prolonged exposure may even cause sun burns as well as skin cancers.


Note : (1) Antimicrobial effect of UV sunlight is on account of the exclusive formation of the ‘singlet oxygen in the cytoplasm’.

(2) Microwaves (in the microwave oven) do not exhibit any direct effect on the microbes, but kill them indirectly by heating the food stuff.


A comprehensive summary of the various physical methods invariably utilized for the effective control of the microbial growth has been duly recorded in Table : 7.3.


Table : 7.3. Comprehensive Summary of Various Physical Methods Utilized for the Effective Control of Microbial Growth


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