Rhizosphere - Microflora, Significance and Rhizosphere effect

The region where the soil and roots make contact is called rhizosphereThe microbial population of rhizosphere is much higher than that of root free soil.

  • Bacterial growth in rhizosphere is enhanced by nutritional substance like vitamins, amino-acids, etc. released from the plants.
  • The roots of higher plants are surrounded by both living as well as non-living environment.
  • The organic and inorganic substances of soil make up the non-living environment.
  • The organism that are present in the soil constitute the living environment.
  • Rhizosphere is the region of soil immediately surrounding the roots of plant.
  • Rhizosphere is divided into two general areas:
  1. Inner rhizosphere : Inner rhizosphere is the area on the root surface.
  2. Outer rhizosphere : Outer rhizosphere is the area near the root surface.
  • The inner rhizosphere has a larger microbial population than the outer rhizosphere as in this region the Interaction between microorganism and roots are most pronounced.
  • The root surface and its adhering soil is sometimes termed the rhizoplane.

Microflora of rhizosphere :

  • The rhizosphere is a highly favourable habitat for proliferation and metabolism of numerous microbial types.
  • The numbers and types of microorganisms differ in case of rhizosphere as compared to root free soil.
  • For microscopic studies of rhizosphere various modifications of Buried slide method of Rossi and Cholodny have been introduced.
  •  In this method the glass slide is Inserted into the soil in such a way that the microbes ultimately grows up along the surface of glass.
  •  The slide is then removed, stained and examined for various types of microorganism.
  • The biochemical techniques used in rhizosphere investigation are numerous, and they are designed to measure a specific change brought about by the plant or by the microflora.
  • Most of the rhizosphere bacteria are saprophytes, some live on the root surface others penetrate the roots.
  • The most abundant bacteria found in rhizosphere are gram-negative rods, gram-positive rods, cocci and spore-formers are less common rhizosphere bacteria.
  • Pseudomonas and Achromogenous spp. are most frequently found bacteria in rhizosphere followed by Agrobacterium.
  • The other bacterial genera found to occur in rhizosphere Include Arthrobacter, Mycoplana, Brevibacterium, Flavobacterium, Serratia, Sarcina, Alginomonas, Bacillus and Mycobacterium.
  • As there is a very high bacterial density (10⁹ celle or more/gm) in rhizosphere there is a high degree of microbial competitions. Fast growing organisms are favoured.

The rhizosphere is influenced by a number of factors:

  • Bacterial count increases in samples taken progressively closer to the plant tissues.
  • Depth of sampling.
  • Different plant species often establish different microflora.
  • The age of the plant also alter the underground part and stage of maturity controls the magnitude of relationship.
  • The stage of maturity controls the magnitude of rhizosphere effect.

(ii) Rhizosphere effect :

  Influence of plant roots on the growth and development of microorganisms in the nearby soil is called rhizosphere effect.

  • It is a stimulus which can be measured by finding out the R:S ratio.
  • Rhizosphere effect is normally measured by plating technique.
  • It is always greater for bacteria than any other microorganism.
  • The R:S ratio is defined as the ratio of microbial numbers/unit weight rhizosphere soil to the population in a unit weight of the adjacent non- rhizosphere control soil.

  • As the distance from rhizosphere increases the R:S ratio decreases.

Influence of plants on rhizosphere microbes :

  • The rhizosphere represents tremendous complex biological system owing to the following chemical activities of plants.
  • Plant roots release a wide variety of materials to their surrounding soil like alcohols, ethylene, sugars, amino-acids, organic acids, vitamins, nucleotides, polysaccharides, enzymes, etc. 
  • These materials favour the growth of microorganisms.
  • Death of plant roots produce a rich source of carbohydrates and their derivatives, which support the growth of many useful bacteria around the plant roots, e.g., nitrogen fixing bacteria, sulfur oxidising bacteria, etc.

Influence of microorganisms :

    Microbial population in the rhizosphere which produces beneficial effects on plant growth are described as Plant Growth Promoting Rhizobia (PGPR). They are a wide range of root colonizing bacteria which promote plant growth.
 Microbial population in the rhizosphere benefit the plants in the following ways :
  • Some bacteria and fungi make N₂ available to plants as nitrates or inorganic form.
  • Sulfur oxidising bacteria make sulfur available as sulfates.
  • Heterotrophic metabolism makes carbon available as CO₂, for photosynthesis.
  • High concentration of CO₂ in the rhizosphere also increases solubility and availability of calcium, and thereby increase calcium uptake by plant roots.
  • Some microorganisms (phosphate solubilisers) also produce acids which convert insoluble rock phosphate to the soluble form and make it available as nutrient.
  • Some bacteria synthesize phyto hormones like auxins, gibberellins, etc. which stimulates the plant growth.
  • Some microorganisms can remove hydrogen sulfide (H₂S) which is toxic to the plant roots.
  • Rhizosphere fungi and bacteria produce siderophores, low molecular weight organic molecules that are able to complex with ferric iron and supply it to the plant cells.
  • Similarly, organic chelating agents produced by microorganisms make manganese compounds available to the plants.
  • Azotobacter and other bacterial spp. produce antifungal substances which protect the plant against microbial invasion.
  • Agarobacter, Rhizobium, Corynebacterium etc. produce cytochyanins which maintains plant growth.
  • Thus, the microflora of rhizosphere is very beneficial to plants.
  • The rhizosphere is the most active factory for transformation of element into living constituents and returning essential elements to soil upon death and decomposition of microorganisms.
  • The hydrolytic forms of microorganisms like cellulose digester transform the plant material into humus, glucose and other valuable food material which can be used both by plant as well as microorganisms.
  • A very heavy growth of microorganisms absorb, nitrogen, sulfur, phosphorus, potassium and other elements in soluble form which might otherwise be removed from the soil by rain and drainage.
  • Thus, higher plants act as a food manufacturer and storage house for microorganisms of soil rhizosphere and microorganisms act as collectors, processors and treasurers of food for higher plants.
Phyllosphere : The environment of the aerial portion of plant is described as phyllosphere.
  • It includes a variety of microbes, including bacteria, molds, yeast and cyanobacteria.
  • The most common phyllosphere bacteria include Pseudomonas, Erwinia, Proteus and Spingomonas spp. most of these microbes exhibit mutualistic association with plants.

Types of Microbial Interactions in Soil : Mutualism, Commensalism, Amensalism, Compitition, Parasitism, Predation & Neutral association

  The microorganisms that inhabit soil exhibit many different types of association or interaction. 

Types of Microbial Interactions

There are three types of Microbial Interactions, found  in soil.
1. Positive interaction :
2. Negative interaction :
- Ammensalism (antagonism),
- Competition.
- Parasitism,
- Predation,
3. Neutral association :

1]. Positive Associations :

(a) Mutualism :

  • This is a symbiotic association where both the partners are benefited.
  • The manner in which the benefit is derived varies.

* Synergism :

  • It is the mutualistic association where both the partners derive benefit from the association.
  • The association is not obligatory.
  • Both populations are capable of surviving Independently, although each gains advantage from the relationship.

* Syntrophism :

  •  It is a type of synergism where two species supply each other's nutritional needs, such as vitamins, amino acids, etc. For example,
  • Association between Enterococcus faecalis and Lactobacillus arabinosus.
  • L. arabinosus requires phenylalanine for growth, which is produced by E. faecalis.
  • E. faecalis requires folic acid which is produced by L. arabinosus.
  • In minimal medium both populations can grow together, but neither can grow alone.

* Rhizosphere effect :

  •   The region where roots and soil make contact is called the rhizosphere.
  • It is a synergistic interaction between microorganisms and plants.
  • From the association both the partners derive nutritional advantage.
  • Association between Thiobacillus ferroxidans and Beljerinckia lacticogenes in medium which lacks carbon and nitrogen sources.
  • T. ferroxidans can fix CO₂ and B. lacticogenes can fix N₂ , thus both can grow in minimal medium devoid of C and N source.
  • This association is useful in bioleaching of copper from its ore.
  • Pure culture of T. ferroxidans can extract 30% of copper from ore.
  • B. lacticogenes can extract 10% of copper from the ore. Both the organisms in association can leach upto 70% copper from its ore.

* Lichens :

  •  Lichens represent mutualistic association between heterotrophic fungi and photosynthetic algae or cyanobacteria. They are usually found on rock surfaces.
  • Algae being photoautotrophic utilise light energy and atmospheric CO₂ to produce organic matter.
  • In some lichens the cyanobacterial partner can also fix atmospheric N₂. Fungi are benefited by getting nutrients produced by algal partners.
  • Fungi on the other hand provide protection and produce enzymes that solubilize rock minerals making essential nutrients available for both the partners.

* Mycorrhizae :

  •   This is an intimate association between plant roots and fungi, where the latter serves as additional roots to acquire nutrients.
  • A mycorrhiza (fungus -root) is a type of endophytic, biotrophic, mutualistic symbiosis found in many natural ecosystems.
  • Several types of mycorrhizal associations are differentiated on the basis of degree of interaction between plant roots and fungi.
  • For example, association between basidiomycetes and roots of forest trees.
  • An (arbuscular mycorrhizal) association occurs in plants belonging to the families: Amaranthaceae, Pinaceae, Betulaceae, Cruciferaceae, etc.

Ectomycorhizae :

  • The fungi penetrates the outermost layers of tree roots and grows on the outer surface of the root. The fungal mycelium forms a sheath around the root of plants.
  • In this association fungi obtain nutrients from plants, and in return it gets water and minerals from the soil through fungi.
  • Most of these fungi cannot be cultivated in absence of plants.
  • The plant growth is adversely affected in absence of fungi.

Endomycorrhizae :

  • In this association fungi grow within the cells of plant roots.
  • Sometimes the fungi form branch like structure or specialized inclusions called vesicles and arbuscules inside the plant cells and so, are called vesicular arbuscular mycorrhizae (VAM).
  • VAM fungi play an important role in increasing plant growth by increasing supply of phosphorous to host plant. Also make the plant more resistant to plant diseases.
  • These arbusculars are digested by the plant cells and the nutrients released from the fungi are used by the plants.
  • The fungi in turn obtain nutrients from the plant tissues.
Schematic diagram of Arbuscular Mycorrhizal Fungi with plant root and its hyphal extension in soil

(b) Commensalism

  •   It is an association between organism in which one partner benefits, while the other partner is not affected.
  1. This occurs in soil with respect to degradation of cellulose and lignin. e.g., association occurs between fungi and bacteria in soil.
  • The cellulose degrading fungi degrades cellulose, produce glucose and organic acids which is used by bacteria for their growth.
  • Thus bacteria are benefited by the association and fungi are not affected.
  • Commensalism also exists when a mixed culture of organisms cause degradation of complex molecules which cannot be done by Individual organism.
  •  E.g.,  pure cultures of microorganisms cannot degrade lignin in laboratory, but the mixed microbial flora can easily degrade lignin forest soil.
  • Many commensal relationships are based on the production of growth factors.
  • Many nutritionally fastidious bacteria in soil often depend on growth factors such as vitamins and amino acids released from other organisms.

2].  Negative associations :

(a) Amensalism / Antagonism :

  •   This is an association where one partner inhibits other organism and thereby gains advantage from the association.
  • The process is called antagonism. The organisms are called antagonist.
  • Their presence in soil is very important, because they produce certain inhibitory substances or antibiotics.
  • These products affect the growth or survival of other organisms e.g.,
  •  Antagonism between bacteria and fungi. Staphylococus aureus produces a diffusable antifungal material that causes distortion and hyphal swellings of Aspergillus terreus.
  • Pseudomonas aeruginosa produces pigment which Inhibits germination of Aspergillus spores.
  • Filamentous actimomycetes are antagonistic for many bacteria. They produce antibiotics which inhibit the growth or competitive population in the soil.
  • Certain fungi present in the soil produce cyanide causing toxic effect on other microorganisms.
  • Some algae produce fatty acids with antibacterial effect.
  • Other antagonistic metabolic products include methane, sulfides and other volatile sulfur compounds.
  • Streptomyces and Myxobacteria produce potent lytic enzymes which destroy other cells by digesting their cell wall or other protective surface layers.

(b) Competition :

  • The organisms, which require same nutrients and similar environmental conditions influence each other.
  • •  They compete for nutrients, light, space, oxygen, etc.
  • •  The organisms, which are adapted to the situation will survive and the rest will be inhibited or destroyed.
  • •  Competitive interactions result in ecological separation of closely related organisms.
  • •  Competition may also limit the growth of all organisms as compared to their potential growth.

(c) Parasitism :

  • This is a relationship in which one organism lives Inside or on the surface of other organism at the expense of the other organism.
  • One partner is called parasite and the other is called host.
  • The parasite feeds on the cells, tissues or body fluids of host, hence it is always harmed.
  • All plants, animals and microorganisms can be attacked by microbial parasites.
  •  e.g., Parasitic association between bacteria. Bacterium Bdellovibrio bacteriovorous present in soil and sewage is a parasite of gram-negative bacteria.

(d) Predation :

  • An association in which one species of organism kills and eats another species.
  • The predator ingests the organism which is a prey. Such predator-prey interactions are of very small or short duration.
  •   e.g., soil fungal are predators of nematodes; amoeba is a predator of bacteria.
  • All these negative associations normally control population densities in soil.
  • The soil fungus Artrobotrys conoides produces hyphae that form rings to trap protozoa and nematodes and digest it.
  • Some fungi such as Trichoderma and Lactisaria species can destroy other plant pathogenic soil fungi. Such mycoparasitic fungi are used as biopesticides to control plant diseases.

3]. Neutral Associations :

  • It is the association in which both partner do not exhibit  positive or detrimental effect on each other.
  • This type of association occurs when each partner can utilise different nutrients without producing end products which is inhibitory to other.
  • The two partners do not compete for nutrition even if they are present in low concentrations.   
  • Such a condition may be transitory as conditions or the relationship might change with variation in environmental conditions.

Biofertilizer - Defination, Types of Biofertilizers and Advantages

  Biofertilizers are defined as " biologically active products or microbial inoculants which help plant's growth."
When plant nutrients are available in abundance the soil is said to be fertile. Microorganisms have a great role in increasing soil fertility.
  Biofertilizers either fix atmospheic N₂ or solublilize plant nutrients like insoluble phosphate or stimulate plant growth through synthesis of growth promoting substances.

Depending upon their activities they are classified as follows :

  1. Symbiotic nitrogen fixers
  2. Asymbiotic nitrogen fixers
  3. Bluegreen algae and Azolla fertilizers
  4. Phosphate solubilizing bacteria ( PSB )
  5. PGPR (Plant growth promoting rhizosphere)
  6. Mycorrhizae
  7. Organic fertilizers.

Disadvantages of Chemical Fertilizers :

  • Increased use of chemical fertilizers causes damage to soil physio - chemical properties.
  • It causes environmental problems like leaching carcinogenic compounds and damage to ozone layer (green house effect) by releasing nitrous oxide (N₂O), carbon monoxide, etc..
  • Chemical fertilizers are expensive.

Advantages of Biofertilizers :

  • It is economical.
  • Biologically fixed N₂.
  • Consumes about 25-30 %  less energy.
  • It is ecofriendly.
  • Biologically originated therefore biodegradable.
  • Non - toxic and non carcinogenic.
  • Easy application with less labour.
  • No sophisticated technology involved.
  • Natural, renewable and economical.
  • Crop yields increases by 15-20%.
  • No adverse effects on crops.
  • They improve the physicochemical properties of soil such as pH, structure, texture and water holding capacity of soil.
  • Simple to use without side effects.
  • Thus, the government promotes the use of biofertilizers.

Limitations of Biofertilizers

- Short shelf - life (less than 6 months), and care in storage is essential.
- Not applicable to all crops, due to specificity between a biofertilizer and plants.

1). Symbiotic nitrogen fixing bacteria

  • Rhizobia are gram-negative, aerobic, non-spore forming soil bacteria.
  • They are able to enter into symbiotic relationship with leguminous plant roots, forming root nodules.
  • In the nodules they fix large quantities of N₂, into NH4+ ions which is available to plants for synthesis of amino acids, proteins, etc.
  • Efficient rhizobia for different crops and location which are tolerant to various stresses like drought, temperature, high or low pH, salinity, etc. are therefore isolated and cultivated on large scale.
  • They are added to carriers like peat or lignite to prepare biofertilizer.
  • Generally they are mixed with 10% sugar or jaggery solution and sprinkled on the seeds, before sowing.
  • When the seeds are sown, bacteria grow along with seed growth. germination and form root nodules, enhancing plant growth.
  • Actinomycetes Frankia species can form symbiotic association with many shrubs and fix nitrogen.

2). Asymbiotic nitrogen fixers

  • Azotobacter and Azospirillum ( also Bacillus polymyxa ) when applied to rhizopshere, fix atmospheric N₂ and make it available to crop plants.
  •   Azospirillum colonizes not only roots, but also above ground parts of the plant through symbiosis.

3). Algal fertilizer ( BGA + Azolla)

  • Blue green algae (BGA) and Azolla (an aquatic fern) are useful biofertilizers in lowland paddy farms.
  • Composite cultures of Blue Green algae including the genera Anabaena, Nostoc, Plectonema, etc. have been more effective than single cultures. Azolla harbours the BGA in leaf cavities providing symbiotic association.
  • BGA fixes atmospheric N₂ which is made available to the crop. They also produce growth promoting substances and provide partial tolerance to pesticides. They help reclaim saline and alkaline soils.

4). Phosphate solubilizing bacteria (PSB)

  • Phoshorus is a limiting nutrient in soils.
  • Bacillus megaterium, var. phosphaticum, B. polymyxa Pseudomonas striata, P. rathoenis and Aspregillus awamori convert nonavailable inorganic phosphate, into soluble organic phosphate which can be utilised by crop plants.

5). Plant Growth Promoting Rhizobacteria (PGPR)

  • PGPR includes Pseudomonas fluorescens and P. putida. They are important new biofertilizers.
  • Pseudomonas species produce pseudobactin a siderophore which chelates Iron and makes it unavailable to harmful fungi in rhizosphere.
  • PGPR has resulted in Increase in yields of potato, radish and other crops upto 30-144% .
  • They also produce plant growth promoting compounds, such as indole acetic acid (IAA).

6). Mycorrhiza

  • Mycorrhiza is a symbiotic association with roots of plants and fungi.
  • The fungi enhances absorption of nutrients from soil by plant roots resulting in health plants.
  • Fungi in turn gets nutrients mainly carbohydrates from plant host.
  • There are two types of mycorrhizae : Ectomycorrhiza and Endomycorrhiza.
  • Ectomycorrhizae are found on the roots of plants and the fungal growth surrounds the root surface. They belong to class Basidiomycetes, Zygomycetes and Ascomycetes.
  • The fungi absorbs nitrogen, phosphorus, potassium and calcium from soil and make it available to plants.
  • They also convert complex organic molecules into simpler available forms.
  • They also protect the roots from pathogens and produce growth promoting substances.
  • Endomycorrhizae are found in the roots of most fruits and horticultural crops.
  • They help the plants in obtaining phosphorus.
  • They also produce growth promoting substances and offer resistance against pathogens.
  • Mycorrhizal biofertilizers have been used for fruit trees such as mango, papaya, tamarind, vegetables and rubber.
  • AM (Arbuscular mycorrhiza) biofertilizers are obtained by growing the fungi using a suitable host plant growing in soil, or sand or its mixture. AM fungi are obligate symbionts and they cannot be cultivated in laboratory media, which is a major limitation for its use and inoculum development.

7). Organic fertilizers :

  •  Organic fertilizers include animal dung, animal urine, bone meals, slaughter house wastes, crop residues, oil cakes, urban garbage, sewage/sludge, sea - weed extracts, etc.
  • All these compounds can be biodegraded and mineralised by soil microbes and the soil fertility can be increased.
  • Humic acid, fulvic acid, etc. are also used in organic fertilizers to improve soil texture and water - holding capacity.

Differences between Prokaryotes and Eukaryotes cell

  All organisms possess cellular organization and made up of one or more cells. The cellular structure is essential in performing various life processes. Each cell can be considered as the structural and functional unit of life.

  All cells possess somewhat similar elemental and molecular constituents. They differ in their structural and ultra structural organization. Based on the differences in their structure, they are of two types prokaryotic cell and eukaryotic cell, and the organisms having such cells are respectively called prokaryotes and eukaryotes.

Prokaryotes vs Eukaryotes
Cell structure of Prokaryotes and Eukaryotes

1 . Prokaryotes -
  Pro means primitive and karyon means nucleus. So basically, the word “prokaryote” means “before nucleus.” 
  Prokaryotes Include those organisms that are simple in cellular organization meaning the cells lack membrane enclosed organelles and possess a poorly defined nucleus, referred to as nuclear material which is also known as nucleoid.
  Prokaryotic nucleus consists of only chromosomal DNA which are present in cytoplasm. Its nucleus is without nuclear membrane and nucleolus.
   Prokaryotic cellular organization does not possess Internal membrane bound cell organelles such as mitochondria, Golgi body, plastids, endoplasmic reticulum etc. They do not possess any differentiated cells or tissues.
  Prokaryotic cells are a lot smaller than eukaryotic cells and have a simpler structure. This simple structure is actually a good thing for prokaryotes, because it allows them to reproduce very quickly and very effectively. 
   Prokaryotes Include two groups of organisms - Bacteria and Achaea.

2. Eukaryotes -
Eu means true or real and karyon means nucleus. So in essence the word “eukaryote” means “true nucleus.”
  Eukaryotes include those organisms that have well developed cellular organization meaning the cells possess membrane enclosed organelles and highly developed nucleus ( membrane - bound ) with nucleolus.
  Eukaryotic cells are larger and much more complicated than prokaryotic cells.

Table : Comparison of Prokaryotes and Eukaryotes
Feature Prokaryotes.                           Eukaryotes 
 Types of Nucleous - Not true membrane bound nucleous.
- Primitive
- It is called nucleoid or chromatin material.                       
 - True membrane bound Nucleous
 Nuclear membrane and nucleous - Absent  - Present
 Chromosome  - Usually, one circular chromosome.                               - More than one linear chromosome occur in the nucleus
 DNA  - Not complexed with proteins (histones).                                       - Complexed with basic proteins called histones.
 Plasmids - verry common  - Rare or absent
 Arrangement of 
 - Linear  - Exons and Introns occur on chromosome
 Cell wall - Peptidoglycan occurs as major unit in cell wall.     
- archaea possess pseudomurein.                                                      
- Peptidoglycan is absent
- May lack cell wall or made up of either cellulose, chitin, keratin, etc
 Cytoskeleton  - Rudimentary  - cytoskeleton consist of microfilaments, intermediate filaments and microtubules.
 Ribosome  - Smaller (70S)  - Larger (80S)
 Gas vesicles  - Present   - Not observed
 Flagelia  - Submicroscopic in size. Consist of a single protein (Flagellin) filament.
- Some possess axial filaments. 
 - Microscopic in size. Membrane bound complex consist of multiple microtubules.
- Some possess cilia.
 Cell Size  - 0.2-2 μm x 2-8 μm  - Diameter greater than 5 μm.
 Permeability of nuclear membrane  - not present  - selectively permeable
Chloroplast  - Absent   - Present in plants
 Mitochondria  - Absent  - Present in animals and fungi
 Lysosomes and peroxisomes   - Absent  - Present
 Genetic recombination  - Partial, undirectional transfers DNA.  - Meiosis and fusion of gametes
 Mode of cell division  - Binary Fission  - Mitosis occurs in somatic (body) cells and meiosis occurs during formation of gametes.
 Metabolism  - Exhibit a great diversity of metabolic pathways.
- Some are strictly or facultative  anaerobic.
 - Follow a common pattern. Glycolysis occurs commonly in most organisms.
- Mainly aerobic respiration, very few are anaerobic.
 Occurrence  - Occurs in diverse ecosystems that provide nutrient.  - Found in diverse habitats as per availability of favourable conditions.
 Phylogenetic groups included  - Domain : Bacteria and Archaea  - Domain - Eukarya, includes Protista, animalia, plantae, fungi

Defferences between Prokaryotic and Eukaryotic cell

• Prokaryotic cells are the oldest type of cell. They are small and relatively simple.
  Eukaryotic cells evolved from prokaryotic cells later. They are larger and much more complex internally.

• Prokaryotes lack both a nucleus and membrane-bound organelles.
  Eukaryotic cells have a nucleus, and also contain organelles.

• Prokaryotes are single-celled organisms.
   Eukaryotes can be either single-celled or multicellular.

• The DNA of a prokaryote is usually organized as a single, circular chromosome, while the DNA of a eukaryote is organized as linear chromosomes. 

Physical methods of Microbial Growth Control

Microbial control by Physical Methods & Agents

  A variety of physical agents and methods can be used for microbial control. Of these, high temperature, radiation and filtration are the agents of choice for achieving sterilization of the objects and environment in laboratories, pharmaceuticals, fermentation industries etc.

Microbial control by use of high temperature

Use of high temperature is one of the most commonly used method for control of microorganisms. It is used as a sterilizing agent obtain total destruction of micro-organisms from the materials. 

  High temperature causes inhibition of microbtal growth by its ability to denature cellular proteins and enzymes as well as other thermo labile substances, Their inactivation kills the organisms.

  The killing effect of heat and sensitivity of organisms to high temperature can be expressed by determination of thermal death point. thermal death time and decimal reduction rate.

Thermal death point -

  The temperature at which all organisms from the substance are killed within 10 minutes is called thermal death point. Resistant organisms possess higher TDP.

Thermal death time and decimal reduction rate -

  The shortest amount of time required to kill all organisms from a given substance under prescribed set of conditions at a given temperature is called thermal death time.

  TDT also indicates degree of temperature resistance of organisms. The death of organisms at high temperature is logarithmic and reduction in microbial number occurs by a specific factor. This is referred to a decimal reduction rate.

   Theoretically 100 % death of organisms is never possible at a given temperature at any time. There Is always a probability of survival of organisms. This rate of reduction in number of microorganisms is also referred to as D value or (del) factor. The time required for reduction of microbial number by one log value is referred to as D value.

              ∇ = K ln N2/N1
∇ = del factor
K = Constant indicating set of conditions
N1 = Initial number of micro-organisms
N2 = Final number of micro-organisms

The graphical presentation of decimal reduction rate is shown in figure

The graph indicates concept of decimal reduction rate and D value.

Types of heat used

  Heat can be applied for the control of micro-organisms in three manners.
1. Direct heat
2. Dry heat
3. Moist heat

1. Control by direct heat

  Application of direct heat by exposing materials to flames causes incineration of the microorganisms present on the surface of materials. It can be achieved by ......
a). Flaming the material by passing it through burner flame.
b). Exposing the materials to infrared radiation. Infrared radiations Increase the temperature of the surface of materials and cause the effect similar to direct heat.

Limitation of direct heat sterilization :
1. The method causes destruction of microorganisms only from the surface and not those, which occur beneath the surface.

2. Only the materials, which can withstand direct flaming. can be sterilized by this method.

Application of direct heat :
Materials like inoculation loops. slides, scalpel, spatula etc. can be sterilized by this method.

2. Control by dry heat

  Another common mode of application of high temperature for control of microorganisms is use of dry heat or hot air. Dry heat kills microorganisms by causing Irreversible denaturation of cellular proteins by oxidation of proteins.

   Various devices are used for control of micro-organisms from materials based on this principle. Hot air ovens are the most common amongst them. The temperature required for obtaining sterilization of materials by this method is 150°C - 200°C for about two hours.

Limitations of dry heat sterilization :
   Dry heat has low penetrating power as well as less effectivity. Therefore, high temperature is to be used for long time achieve sterilization. Therefore, only those materials, capable of withstanding these conditions can be sterilized by this mode.

Application of dry heat sterilization :
This method of steriltzation applied for sterilization of laboratory glass wares, dry powders or similar such materials.

3. Control by moist heat

  Application of moist heat as sterilizing agent is one of the most common approaches to achieve sterilization. It is achieved by exposing materials to steam.

  Moist heat causes death of microorganisms by denaturing cellular proteins. It causes coagulation of cellular proteins. Compared to dry heat, moist heat has more penetration power and effectivity. Therefore, to achieve sterilization, relatively steam at lower temperatures and application for shorter durations is required.

  Amongst all forms of microorganisms, spores of bacteria are most resistant. They are killed by steam at 121.6°C within 10 to 15 minutes, whereas similar results are obtained by dry heat within 2 hours.

Modes of application of moist heat :

Moist heat sterilization can be achieved in three different modes of treatments.
a). At temperature below 100°C. 
b). At 100 C.
c). At temperature above 100°C.

a). Sterilization at temperature below 100° C -

  This method is used when the materials to be sterilized are thermo labile and cannot withstand higher temperature of treatment. Sterilization by this method involves application of the principle of fractional sterilization or tyndallization.

• Tyndallization -

This method of control was initially evolved by Tyndall. Therefore, It is called tyndallization. It is also known as fractional sterilization or intermittent sterilization due to its mode of application. The method does not involve destruction of all the organisms at a time, but they are killed intermittently or fractionally in different phases.

• Method of tyndallization -

  Tyndallization involves treatment of the material at 70°C - 80°C for about 15 minutes in water bath for three successive days, to achieve total destruction of microorganisms. This treatment achieves sterilization as under.

1). When the material is exposed to 70°C - 80°C for 15 minutes in water bath on the first day, It will kill all vegetative cells from the material but not spores. This is because, spores are heat resistant.

2). Now when the material is incubated for 24 hours after first exposure to heat, the spores will germinate and get converted into vegetative forms. These spores are then killed by exposure of material to 70°C - 80°C again for 15 minutes in water bath.

3). Still, there is a possibility that some spores might not have germinated. They may survive during this second heat treatment. To ensure the destruction of these surviving spores, the material is again incubated at 37°C for 24 hours. 

  During this period, the surviving spores will also germinate to form vegetative cells. They are then killed by again heating the material at 70°C to 80°C for 15 minutes in water bath on the third day. Thus, the method Involves killing of all organisms from the objects by use of relatively low temperature.

• Use of tyndallization -

  Inspissator is the most commonly used instrument for sterilization of materials by tyndallization. The method is used to achieve sterilization of various thermo labile materials, such as egg media, serum and serum containing media etc.

b). Sterilization at of temperature 100°C -

Sterilization at 100°C temperature is achieved by use of boiling water bath or Arnold sterilizer. The method is used for sterilization of materials and objects which resist boiling. Since boiling cannot destroy bacterial spores, principle of tyndallization is employed to achieve total destruction of all forms of micro- organisms.

c). Sterilization at temperature above 100°C -

  Steam under pressure is used to achieve sterilization by moist heat at temperature above 100°C. Temperature of steam is directly proportional to pressure as shown in the table.

Table : Relationship between steam pressure and temperature
Steam pressure
Temperature °C
    0     100.0
5 109.0 
10 115.0 
15  121.6 
 20 126.5 

Steam under pressure, has greater penetration power and effectivity. Hence the death of organisms is rapid at higher steam pressure and temperature. Usually, steam at 15 Ibs/sq in pressure is recommended for sterilization of objects. At this treatment, even spores of bacteria are killed, Autoclave or steam sterilizer is the most commonly used instrument, used in laboratory, working on this principle.

Application of moist heat sterilization :

   Materials, which can withstand high stem pressure and temperature, can only be sterilized by use of this method. The method is suitable for sterilization of routine bacteriological media, glassware, surgical instruments, gauges, clothes ete.

Control by use of low temperature

  Decrease in temperature of incubation results in the decrease in the overall rate in the cellular metabolism and hence the rate of growth. Therefore, low temperature is widely used for the control of microblal activities in the materials, especially for preservation of foods, food products and beverages. 

 Control of organisms, using low temperature can be achieved at
a). refrigeration temperature, i.e. at 4°C to 7°C
b). freezing temperature i.e. at temperature below 0°C.

Control at refrigeration temperature -

  The method is most commonly used for preservation of foods and food products in households and culture preservation. Low temperature causes retardation in microbial activities and exerts bacteriostatic effect.

Control at freezing temperature -

  Exposure of organisms at subzero temperature results in to freezing and formation of ice crystals within the cell. Hence when organisms are expressed to subzero temperature cellular water content gets frozen. This result in -
a). Mechanical damage to cells and some of the organisms are killed.
b). Arrest of cellular metabolism and hence growth of organisms.

   However organisms can survive for long time even the low temperature. Hence, use of freezing temperature achieves microbial control by inhibiting growth rather than killing of organisms.

Microbial Growth Control by Desiccation

  Removal of water is called desiccation. Desiccation of microblal cells and their environment stops microbial growth and activities. This is because Water required for activity of cellular enzymes as well as growth is not available.

   This is one of the most widely used methods for microblal control of food spoilage as well as the preservation of cultures during freeze drying method.

   A number of factors decide the efficiency of microbial control by desiccation. They include:
a). The type of micro-organisms.  
b). The type of substances on which organisms are dried.
c). Physical conditions to which organisms are exposed to after desiccation such as light. temperature, humidity etc.
  Usually, gram-negative bacteria are most musceptible to desiccation.

  They include gram-negative cocci, spirochetes etc. Gram- positive cocci and rods are more resistant to desiccation.

  Spore formers as well as capsulated organisms can withstand conditions of desiccation for long duration. Mycobacteria are one of the classical examples of the organisms which can survive after desiccation for exceptionally long durations. This is because of the layer of myclic acid in their cell wall, which protects cytoplasm from desiccation. Fungal spores, conidia are also resistant to desiccation.

Control by use of osmotic pressure

  The difference in the concentration of solutions across semi permeable membrane causes passage of solvent from lower solute concentration to higher solute concentration tiIl the equilibrium is achieved. This phenomenon is called osmosis. The pressure resulting due to the passage of solvent on the membrane is called osmotic pressure.

  When the cells are exposed to high osmotic conditions (high concentrations), water from cells come out resulting into plasmolysis of cells. This results into desiccation of cells that inhibit metabolic activities, and cause control of microbial growth.

  Similarly, when cells are exposed to low osmotic conditions, water from outside enter the cell causing plasmoptyses. The cells get ruptured as a result of this phenomenon. Thus also helps in achieving microbial control.

   The practical application of this method is in the control of food spoilage where foods can be preserved using high brine solution syrups etc., in which development of high osmotic conditions exert control.

Microbial Control by Sonication

Bacteria can be disrupted by using sonic and ultrasonic waves. Sound waves with a frequency of 720000 cycles/sec. are more effective. They can be achieved by use of electrically vibrating needle or disc.

Mode of action :

   When the bacteria, suspended in liquid, are exposed to ultrasonication, the sound waves cause cellular damage by cavitation. The passage of sound through liquid causes formation of cavities. The cavities grow in size, until they collapse violently. 

  This results in to disintegration of cells. Thus, sonic waves kill the organisms. Gram-negative bacteria are most susceptible to sonication as compared to cocci and gram-positive rods.

Microbial Control by Radiations

  Radiations, which have shorter wavelength, are called energy radiations. They possess a higher quantum of energy. These radiations are lethal to the organisms.

  Therefore, these radiations are used for the control of microorganisms. These high energy radiations are of different types:
- Ultraviolet rays,
- X-rays,
- Gamma rays and
- Cathode rays.
  Sterilization by use of radiation Is also known as cold sterilization.

Ultraviolet rays

The light radiations with wavelength between 2000 A° to 4000 A° are called ultraviolet rays. They are able to kill organisms due to their ability to -
a). denatures cellular macromolecules.
b). cause ionization of the cellular molecules.

Denaturation of cellular macromolecules by UV rays :

  Of the different macromolecules of cells, nucleic acids and proteins are the most sensitive molecule which gets denatured by UV rays. This is mainly due to the absorption of UV light by nucleic acids and proteins.

- Effect of UV on DNA

   DNA can absorb maximum UV light at 2654 A° λ. This results into formation of pyrimidine dimers in to DNA. Formation of the pyrimidine dimers in the DNA affects the DNA structure.

  This, in turn affect the normal functioning of DNA and interfere with DNA transcription and its replication. This ultimately influences viability of the cells, resulting into death of cells

- Effect of UV on RNA and proteins

  RNA and protein molecules can also absorb maximum UV light at 260 nm. This results into alteration in the structure and conformation of cellular RNA and proteins, which in turn, contribute to the killing of cells by UV rays.

Ionizing activity of UV rays :

   UV rays. being energy radiations, also possess some ionizing effect in the cell. They cause lonization of water molecules present in cell, which generate toxic effect.

Use of UV in microbial control :

UV rays are widely used for obtaining sterilization in closed room areas and inoculation chambers. It is also widely used in operation theaters, aseptic areas of pharmaceutical industries etc. to achieve sterilization.

Ionizing radiations

   Electromagnetie radiations with extremely short wavelength possess extremely high energy. Such radiations are therefore able to cause lonization of the target molecules. Therefore, these radiations are called ionizing radiations. These radiations are lethal to the organisms. lonizing radiations include X-rays, gamma rays and cathode rays.

- X-rays

X-rays possess wavelength, smaller than UV rays. They were first discovered by Rontgen. Hence, they are also called Rontgen rays.

Mode of action :
  Since these radiations possess very short wavelength. they possess a high penetration power as well as lonizing property. They act by -
1. Damaging DNA and RNA due to their ability to break phosphodiester bond in the nucleie acids.
2. Ionizing various molecules of cytoplasm. especially water, by forming OH- ions, which are toxic.

  Though, X-rays possess antimicroblal activity, their practical application is rare. This is because -
1. They are expensive.
2. Their controlled use is difficult because of their ability to disperse in all direction.

- Gamma rays
These are also high energy radiations, emitted by radioactive isotopes. Usually radio isotope Co⁶⁰ is used for this purpose. These radiations also act by caustng lontzation of almost all cellular molecules, especially water, by forming hydroxyl ions, having toxie effect.

Application :
   Gamma rays are widely used commercially for sterilizing various materials in packed conditions. These include plastic injection syringes, needles, Petri dishes and other surgical materials. food and food products etc. The commercial application of gamma rays for obtaining the packed materials has become possible due to their high killing as well as penetration power.

- Cathode rays
  Cathode rays have the smallest wavelength (0.05 A°). Therefore, they possess maximum energy component and maximum lonizing ability. Cathode rays can be generated by creating a high voltage difference between cathode and anode potentials. A very high voltage of electric current is gven to a cathode, which emits electrons. These electrons are called cathode rays.

Mode of action :
   The cathode rays act in the same manner, as other ionizing radiations act. They cause ionization of call cellular molecules leading to the death of cells.
   Though cathode rays possess a high killing power than all other ionizing radiations, their use in microblal control is limited because of their high cost. However, they can be applied to obtain sterilization of surgical materials. drugs etc.

Microbial control by filtration

  Physicall removal of organisms from materials / system is one of the most commonly employed methods to achieve microbial control. Use of bacteriological filters for the purpose is a widely used technic.

Principle of microbial control by filtration-

  Passage of liquid or gas through a filter having a specific pore size has a capability to retain particles of larger size. Hence employing the use of a filter having pore retain small enough to micro-organisms of certain size causes removal of these organisms from the liquid or gas treated with.

   In addition, certain filters cause removal of organisms by adsorption also. Charged surface of these filters ald in removal of negatively charged bacteria from the system by causing their adsorption on the filter surface.

Types of filters used for microbial control

A variety of filters made of different materials are employed for the removal of micro-organisms from liquids to be sterilized. These filters are made of materials like clay, paper, asbestos, glass, diatomaceous earth, cellulose acetate etc.

   These filters a designed to have pores with diameters small enough to retain bacteria. The earliest filters of these types are Chamberland filter, developed by Chamberland at Pasture's laboratory. Following are the different types of bacteriological filters.

• Porcelain filters

  These are made of unglazed porcelain. They are available in different grades. These were the earliest filters to be developed by Chamberland. Therefore, these filters are also known as Chamberland filters.

• Berkfeld filters

  These filters are made from a mixture of diatomaceous earth, asbestos, plaster of Paris and water. They are made in form of a hollow candle with graded pore size. The fluid to be filtered is forced by pressure or suction from outside to inside or vice versa, allowing microorganisms to be retained on the fiter surface. These filters are also commonly referred to as candle filters.

• Sintered glass filters

  These filters are made from finely ground glass beads. The glass beads are allowed to fuse to adhere sufficiently to allow the beads to adhere each other leaving pores between them. These are available in form of disc, fused with glass funnel.

• Asbestos filters

   A sheet of asbestos can also provide a fltering surface and hence it can be used as filter. Seitz filter is one of these types of filters. In this filter, asbestos sheet is clamped between two perforated metal dises with a metal funnel assembly.

• Membrane filters

   These filters are also known as molecular sieve filters. They are manufactured from a variety of polymeric materials such as cellulose diacetate, cellulose nitrate, polycarbonate, polyester etc. These niters were first introduced by Millipore inc. as milipore filters.

  The membrane filters are available with destred grade of pore size ranging from 0.015 nm to 12 nm. Specially designed assembly is used to hold membrane filters for sterilization of liquids.

Preparation of bacteriological filters for the use -

  A special care has to be taken before a bacteriological filter is used for the sterilization of liquids.
1). The filter should be properly cleaned before use. If asbestos filter of membrane filters are used, new filter sheet is required for the purpose.
2). The filter assembly is properly sterilized by autoclaving before its use.
3). Usually vacuum flask, connected to vacuum pump is used to collect filtered liquid. Vacuum creates a negative pressure in the flask and helps in rapid filtration.

Applications of bacteriological filters -

  Bacteriological filters are employed to steriltze liquids like water, broth media, antibiotics, vitamin and amino acid solutions, hyper immune sera etc. In general. the liquids, which are thermo labile and unable to get sterilized by heat, are sterilized by use of bacteriological filters.

High Efficiency Particulate Air (HEPA) filters :

  HEPA filters are made from cellulose acetate, pleated around aluminum foil. They are available in various sizes for different applications. These filters have ability to remove 99.97% of the particles having size 0.3 nm or more from air. These filters are commonly employed In obtaining clean air environment in pharmaceuticals, laboratories, Industries, hospitals etc.

Laminar air flow system

   Laminar air flow system is one of the commercial application of HEPA filters to obtain sterile environment in a given area. Here, the air is allowed to flow in one direction with uniform velocity in a confined are after its passage to HEPA filters. It removes air bore particles and maintains a clean environment in the area through which air is allowed to flow.

Application :

   Laminar air flow has application not only in obtaining sterile area in inoculation chambers in microbiology laboratories, but has also got wide applications in creating clean room environment. especially in pharmaceuticals. operation theatres, electronic industries, biotechnology laboratories etc.

Principles of Control of Microorganisms & Terms associated with it

Control of micro-organisms means reduction in the number and/or activity of micro-organisms. The main objectives behind the microbial control include :
1. Prevention of occurrence and transmission of diseases and infection.
2. Prevention of cotamination or growth of undesirable organisms, especially during microbiological studies, fermentation processes, analytical methods or processes involving use of microorganisms.
3. Prevention and control of deterioration of materials or spoilage foods of by micro-organisms.

Principles of microbial control :

  Control of micro-organisms can be achieved by meeting with any of the following criteria.
1. Killing of micro-organisms.
2. Removal of micro-organisms.
3. Inhibition of the growth and activities of micro-organisms.

The agents capable of killing the micro-organisms or Inhibiting their growth and activity are called antimicrobial agents.

General mechanisms of antimicrobial action

  Viability of the organisms and their ability to grow depends on the normal structure and function of many vital components of cell, especially cell wall, cell membrane, nucleie acids, proteins and activity of several enzymes performing metabolic pathways.
   The antimicrobial agent effect one or many of these cellular components and influencing viability of organisms. The mode of action of various antimicrobial agents can be generalized as under.

• Damage to cell wall and/or inhibition of cell wall synthesis - 

   Bacterial cell wall is a rigid structure and protects the cell from lysis due to osmotic changes in the environment. Therefore, damage to cell wall, its lysis or inhibition of cell wall synthesis cause lysis of cells and kills bacteria.

• Interference of cell membrane function -

Cell membrane of bacteria is selectively permeable. It controls exit and entry of molecules from and into the cell. Thereby, It controls cytoplasmic concentration. In addition, it possesses various important enzymes, essential for vitality of cell. Agents, able to damage cell membrane, may alter its selective permeability or interfere with its function and hence affect the cell. usually causing Its death.

• Interference with protein - 

   Structure, function and synthesis Proteins are important macromolecules of cell. Many of them are essential for formation of cell structures. Others are essential for cell activity. Hence the agents. which modify physiochemical structure of proteins, change their conformation or inhibit protein synthesis are able to inhibit the growth or kill the organisms.

• Effect on cellular nucleic acids -

   Both the types of nucleic acids, RNA and DNA, play a key role in vitality of cells. RNA participates in protein synthesis. DNA acts as genetic material. Hence alterations in structure of the nucleic acids or inhibition of their synthesis inhibit the organisms.

• Inhibition of enzyme action -

   Enzymes are responsible for all metabolic processes going on within the cell. Therefore, agents capable of Interfering with or inhibiting the activity of enzymes Interfere with metabolism and hence the growth.

Terms associated with the control of micro-organisms

Following terms are commonly used to describe processes and agents used for controlling micro-organisms.

Sterilization :

The process of destroying or removal of all forms of living organisms from the materials is called sterilization. Such objects, free from micro-organisms are called sterile
  The process is employed to remove all the contaminating. unwanted organisms from the materials which may affect cultivation. study or application of micro-organisms. This method is mainly used in fermentation industries and laboratory for sterilize culture media.

Pasteurization :

  The process, in which only harmfulpathogenic organisms are aimed at removal, without affecting quality of material is commonly known as pasteurization. 
This process is normally employed for treatment of milk. food and beverages (bear and wine) to destroy pathogens or harmful contaminants, The term is named so after the inventor of the process, Louis Pasteur.

Disinfection and Disinfectant : 

  Disinfection is the process of destroying infectious pathogenic mlcroorganisms, The agent used for the purpose Is called disinfectant. 
  These agents are usually the chemicals which destroy vegetative growing formsol pathogens. but not necessarily the spores. The term is usually applied when the treatment is applied to Inanimate objects.

Antisepsis and antiseptic :

  The process that prevents sepsis i.e. growth or activity microorganisms is called antisepsis. The agent used for the purpose Is called antiseptic.
   These are also usually the chemical agents that act by killing the micro-organisms or inhibiting their growth. Usually term is used, when the agent is applied to the body.

Sanitization and sanitizer :

  The process in which the microbial number in the environment or materials is reduced to a safe level as Judged by public health standards is called sanitization
  Agents used to achieve sanitization are called sanitizer. Reduction in the number of microorganisms to a safer low level reduces chances of infections and hence helps in maintaining general public health parameters. 
  These agent may be physical or chemical and are applied usually to Inanimate objects such as utensils, vessels and equipments used in dairy and food Industries, restaurants etc. as well as environment such as air and water.

Germicide :

  An agent that is used to kill growing organisms but not necessarily spores is called germicide. Thus, the term is synonymous with disinfectant. But, germicides are used commonly to destroy but all forms of micro-organisms (germs) not only the Infectious organisms.

Microbicide :

  An agent able to kill micro-organisms is called microbicide. They can be categorized in to different types on the basis of the group of micro-organisms they kill. 
  Accordingly they can be grouped as bactericide, viricide, fungicide, algicide, sporicide etc. These agents are usually chemicals.

Bacteriostatic :

  The agent that only inhibits growth of bacteria but does not kill them is called bacteriostatic agent. The process is called bacteriostasis.

Rate of Death of Organisms & Factors Influencing it

  The death of microorganisms, due to the effect of antimicrobials, always occur in a logarithmic manner. An antimicrobtal agent causes death of micro-organisms by killing a specific proportion of organisms present in the object under a given set of conditions.

  Therefore, the decrease in the number of organisms become logarithmic or exponential. For example, a given agent is able to kill 90% of the population under a given set of conditions per unit time the number of survivors will be as under.
10(n), 10 (n-1), 10 (n-2), 10(n-3) and so on
Where, n indicates initial logarithmic value of organisms present in the object / material.

The death curve of organisms exposed to an antimicrobial agent. Curve A shows arithmetic number of organisms surviving per unit volume against time. Curve B shows logarithmic value of organisms surviving per unit volume against time.

This probability of death of micro-organisms is also proportional to the concentration of the antimicrobial chemical agent or intensity of the physical agent. If the concentration of the chemical or intensity of the physical agent is more, rate of death will be more, and vice versa.
   Further, rate of death is also influenced by various other factors such as
- age of organisms,
- physiology of cells,
- types of organisms as well as environmental conditions of treatment.
  The overall death of organisms will also depend on the time of treatment as well as initial microbial load.

Importance of study of kinetics of microbial death
  The study indicates the probability of surviving micro- organisms at the end of treatment. The probability Increases with higher iInitial microbial load. Hence time required to achieve effective sterilization depends on Initial microbial load. Thus the study allows to calculate the time required to achleve effective sterilization.

Factors influencing death of micro-organisms

  Various factors are known to infuence the death of micro-organisms on treatment with antimierobial agents. They include :
1. Type of microorganisms
2. Biological and physiological characteristics of the organisms
3. Initial microbial load
4. Time of treatment
5. pH
6. Viscosity of the material
7. Chemical composition of the material
8. Temperature
9. Concentration or intensity of antimicrobial agent

1. Type of micro-organisms -

  The species of micro-organisms present in the material is one of the important factors that decides rate of death. Sensitivity of an organism to an agent varies with the species. Some species are most sensitive to an agent. Usually spores of bacteria are most resistant to any antimicrobial agent than the vegetative cells.
   Amongst bacteria, usually cocet and gram-positive organisms are found more resistant to an agent than gram-negative organisms.

2. Biological and physiological Characteristics of the organisms - 

   The biological and physiological properties of organisms are also one of the factors that decide effectivity of antimicrobial agents and hence rate of death. In general, old cells as well as resting or dormant organisms are resistant, whereas actively growing young cells are easily killed. This is because of the fact that most agents act by interfering with cell's metabolism, which is most active in actively growing young cells.

3. Initial microbial load -

   If the initial microblal load in the material is high, a more number of organisms is required to be killed. Since, any antimicrobial agent kills organisms in a specific proportion per unit time of treatment, more treatment is required to kill all organisms from the material if initial microbial load is high and vice versa.

4. Time of treatment -

  The organisms present in a material are always killed function of time, when treated with antimicrobial agent. Therefore, if duration of treatment with antimicrobial agent is increased, more organisms will be killed and vice versa.

5. pH -

  pH is inverse log of H ion concentration. It affects the potency of any antimicroblal agent. Usually, the effectivity of most antimicrobial agents increase with increase in H ion concentration of material, i.e. at lower pH. However, It also depends on the property of the agent.
i).  e.g. Heat, radiations etc. are more effective at low pH values.
ii). Effect of certain agents such as penicillin decrease at low pH values as they get inactivated.

6. Viscosity of the material -

   Another physical factor affecting efficiency of an antimicrobial agent is viscosity. High viscosity has a protective effect and hence reduces the death rate of organisms.

7. Chemical composition of material -

  Presence of large amount of organic matter in the material has the protective effect against the antimicrobial agent. It may adsorb the antimicrobial agent and retard its eflectivity.

8. Temperature -

   Temperature of treatment is another important factor which influences death rate of bacteria by antimicrobtal agent. Usually. the effectivity increases with the increase in temperature of treatment.

9. Concentration or intensity of the agent -

   The death rate of bacteria is always Increasing with the increase in the concentration of antimicrobial chemical or intensity of the physical agent.