Chemical Methods of Sterilization and Disinfection

Ethylene oxide. This highly reactive gas (C2H4O) is flammable, toxic, and a
strong mucosal irritant. Ethylene oxide can be used for sterilization at low
temperatures (20–60 8C). The gas has a high penetration capacity and can
even get through some plastic foils. One drawback is that this gas cannot
kill dried microorganisms and requires a relative humidity level of 40–
90% in the sterilizing chamber. Ethylene oxide goes into solution in plastics,
rubber, and similar materials, therefore sterilized items must be allowed to
stand for a longer period to ensure complete desorption.

Aldehydes. Formaldehyde (HCHO) is the most important aldehyde. It can be
used in a special apparatus for gas sterilization. Its main use, however, is in
disinfection. Formaldehyde is a water-soluble gas. Formalin is a 35% solution
of this gas inwater. Formaldehyde irritatesmucosa; skin contactmay result in
inflammations or allergic eczemas. Formaldehyde is a broad-spectrum ger-
micide for bacteria, fungi, and viruses. At higher concentrations, spores
are killed as well. This substance is used to disinfect surfaces and objects
in 0.5–5% solutions. In the past, it was commonly used in gaseous form to
disinfect the air inside rooms (5 g/m3). The mechanism of action of formal-
dehyde is based on protein denaturation.
Another aldehyde used for disinfection purposes is glutaraldehyde.

Alcohols. The types of alcohol used in disinfection are ethanol (80%), propanol
(60%), and isopropanol (70%). Alcohols are quite effective against bacteria and
fungi, less so against viruses. They do not kill bacterial spores. Due to their
rapid action and good skin penetration, the main areas of application of al-
cohols are surgical and hygienic disinfection of the skin and hands. One dis-
advantage is that their effect is not long-lasting (no depot effect). Alcohols
denature proteins.

Phenols. Lister was the first to use phenol (carbolic acid) in medical applica-
tions. Today, phenol derivatives substituted with organic groups and/or halo-
gens (alkylated, arylated, and halogenated phenols), are widely used. One
common feature of phenolic substances is their weak performance against
spores and viruses. Phenols denature proteins. They bind to organicmaterials
to a moderate degree only, making them suitable for disinfection of excreted
materials.

Halogens. Chlorine, iodine, and derivatives of these halogens are suitable for
use as disinfectants. Chlorine and iodine show a generalized microbicidal ef-
fect and also kill spores.
Chlorine denatures proteins by binding to free amino groups; hypochlo-
rous acid (HOCl), on the other hand, is produced in aqueous solutions, then

disintegrates into HCl and 1/2 O2 and thus acts as a powerful oxidant. Chlorine
is used to disinfect drinkingwater and swimming-poolwater (up to 0.5mg/l).
Calcium hypochlorite (chlorinated lime) can be used in nonspecific disinfec-
tion of excretions. Chloramines are organic chlorine compounds that split off
chlorine in aqueous solutions. They are used in cleaning and washing pro-
ducts and to disinfect excretions.
Iodine has qualities similar to those of chlorine. Themost important iodine
preparations are the solutions of iodine and potassiumiodide in alcohol (tinc-
ture of iodine) used to disinfect skin and small wounds. Iodophores are com-
plexes of iodine and surfactants (e.g., polyvinyl pyrrolidone). While iodo-
phores are less irritant to the skin than pure iodine, they are also less effective
as germicides.

Oxidants. This group includes ozone, hydrogen peroxide, potassiumperman-
ganate, and peracetic acid. Their relevant chemical activity is based on the
splitting off of oxygen. Most are used as mild antiseptics to disinfect mucosa,
skin, or wounds.

Surfactants. These substances (also known as surface-active agents, tensides,
or detergents) include anionic, cationic, amphoteric, and nonionic detergent
compounds, of which the cationic and amphoteric types are the most effec-
tive (Fig. 1.8).
The bactericidal effect of these substances is onlymoderate. They have no
effect at all on tuberculosis bacteria (with the exception of amphotensides),
spores, or nonencapsulated viruses. Their efficacy is good against Gram-pos-
itive bacteria, but less so against Gram-negative rods. Their advantages in-
clude low toxicity levels, lack of odor, good skin tolerance, and a cleaning ef-
fect.

Temporary Bridge for Temporary ISBT at Haridwar for 2010 Mahakumbh

As I was going to the Daksha temple today, I saw this construction going on for the temporary bridge for temporary ISBT at Kankhal, Haridwar for the upcoming Mahakumbh 2010. It will help in the diversion of the buses for Bijnor and following cities. Its nice to see that work is being done for improvement of city. Hope there will be a permanent ISBT soon.

Bought new Canon Powershot A480

Today, I bought a Canon powershot A480 camera, after waiting for nearly one year. I bought it from Kantika Digital Colour lab, Haridwar.

Physical Methods of Sterilization and Disinfection

Heat
The application of heat is a simple, cheap and effective method of killing
pathogens. Methods of heat application vary according to the specific appli-
cation.
& Pasteurization. This is the antimicrobial treatment used for foods in li-
quid form (milk):
— Low-temperature pasteurization: 61.5 8C, 30 minutes; 71 8C, 15 seconds.
— High-temperature pasteurization: brief (seconds) of exposure to 80–85 8C
in continuous operation.
— Uperization: heating to 150 8C for 2.5 seconds in a pressurized container
using steam injection.
& Disinfection. Application of temperatures below what would be required
for sterilization. Important: boiling medical instruments, needles, syringes,
etc. does not constitute sterilization! Many bacterial spores are not killed
by this method.
& Dry heat sterilization. The guideline values for hot-air sterilizers are as
follows: 180 8C for 30minutes,160 8C for 120minutes,whereby the objects to
be sterilized must themselves reach these temperatures for the entire pre-
scribed period.
& Moist heat sterilization. Autoclaves charged with saturated, pressurized
steam are used for this purpose:
— 121 8C, 15 minutes, one atmosphere of pressure (total: 202 kPa).
— 134 8C, three minutes, two atmospheres of pressure (total: 303 kPa).
In practical operation, the heating and equalibriating heatup and equalizing
times must be added to these, i.e., the time required for the temperature in
the most inaccessible part of the item(s) to be sterilized to reach sterilization
level.When sterilizing liquids, a cooling time is also required to avoid boiling
point retardation.

The significant heat energy content of steam, which is transferred to the
cooler sterilization itemswhen the steamcondenses on them, explainswhy it
is such an effective pathogen killer. In addition, the proteins of microorgan-
isms are much more readily denatured in a moist environment than under
dry conditions.

Radiation
& Nonionizing radiation. Ultra-violet (UV) rays (280–200 nm) are a type of
nonionizing radiation that is rapidly absorbed by a variety of materials. UV
rays are therefore used only to reduce airborne pathogen counts (surgical
theaters, filling equipment) and for disinfection of smooth surfaces.
& Ionizing radiation. Two types are used:
— Gamma radiation consists of electromagnetic waves produced by nuclear
disintegration (e.g., of radioisotope 60Co).
— Corpuscular radiation consists of electrons produced in generators and
accelerated to raise their energy level.
Radiosterilization equipment is expensive. On a large scale, such systems are
used only to sterilize bandages, suture material, plastic medical items, and
heat-sensitive pharmaceuticals. The required dose depends on the level of
product contamination (bioburden) and on how sensitive the contaminating
microbes are to the radiation. As a rule, a dose of 2.5 !104 Gy (Gray) is con-
sidered sufficient.
One Gy is defined as absorption of the energy quantum one joule (J)
per kg.

Filtration
Liquids and gases can also be sterilized by filtration. Most of the available
filters catch only bacteria and fungi, but with ultrafine filters viruses and
even large molecules can be filtered out as well. With membrane filters, re-
tention takes place through small pores. The best-known type is the mem-
brane filtermade of organic colloids (e.g., cellulose ester). Thesematerials can
be processed to produce thin filter layers with gauged and calibrated pore
sizes. In conventional depth filters, liquids are put through a layer of fibrous
material (e.g., asbestos). The effectiveness of this type of filter is due largely
to the principle of adsorption. Because of possible toxic side effects, they are
now practically obsolete.

Simple Influenza test is not reliable for Swine flu

The experts and doctors dealing with swine flu are emphasizing that the influenza test is not at all reliable for the swine flu virus H1N1. There are some cases where the patient had swine flu, but the influenza test was negative.
The swine flu virus itself has mutated several times by now. So, it is being very difficult to diagnose the problem and that too with Influenza test.

Evolution of Eucaryotic Cell

The profound differences between eucaryotic and procaryotic cells have stimulated much discussion about how the more complex eucaryotic cell arose. Some biologists believe the original ‘protoeucaryote’ was a large, aerobic archaeon or bacterium that formed mitochondria, chloroplasts, and nuclei when it plasma membrane invaginated and enclosed genetic material in a double membrane. Theorganelles could then evolve independently it also is possiblethat a large cyanobacterium lost its cell wall and became phagocytic. subsequently, primitive chloroplasts, mitochondria, and nuclei would be formed by the fusion of thylakoids and endoplasmic reticulum cisternae to enclose specific areas of cytoplasm. By far the most popular theory for the origin of eucaryotic cells is the endosymbiotic theory. In brief, it is supposed that the ancestral procaryotic cell, which may have been an archaeon, lost its cell wall and gained the ability to obtain nutrients by phagocytosing other procaryotes. When photosynthetic cyanobacteria arose, the environment slowly became oxic. If an anaerobic, amoeboid, phaocytic procaryote-possibly already possessing a developed nucleus- engulfed an aerobic bacterial cell and established a permanent symbiotic relationship with it, the host would be better adapted to its increasingly oxic environment. The endosymbiotic aerobic bacterium eventually would developinto the mitochondrion. Similarly, symbiotic association with cyanobacteria could lead to the formation of chloroplasts and photosynthetic eucaryotes. Some have speculated that cilia and flagella might have arisen from the attachment of spirochete bacteria to the surface of eucaryotic cells, much as spirochetes attach to themselves to the surface of motile protozoan Myxotricha paradoxa that grows in the digestive tract of termites. There is evidence to support the endosymbiotic theory. Both mitochondria and chloroplasts resemple bacteria in size and appearance, contain DNA in the form of a closed circle like that of bacteria, and reproduce semiautonomously.Mitochondrial and chloroplast ribosomes resemble procaryotic ribosomes more closely than those in the eucaryotic cytoplasmic matrix.The sequences of the chloroplast and mitochondrial genes for ribosomal RNA and transfer RNA are more similar to bacterial gene sequences than to those of eucaryotic rRNA and tRNA nucltargeles. Finally, there are symbiotic assciations that appear to be bacterial endosymbioss in which distinctive procaryotic characterisics are being lost. for example,the protuzoam flagellate cyanophora paradoxa has photosynthetic organelles called cyanellase with a structure similar to that of cyanobacteria and the remain of peptidoglycan in their walls.their DNA is much smaller than that of cyanobacteria and resembles chloroplast DNA.

Germ Theory Of Disease

The ‘germ theory of disease’ has presented a great stimulus in Microbiology and medicine. Louis Pasteur and Robert Koch (1843 – 1910) were the national heroes. Preventive measures also supported the germ theory. Edward Jenner (1796) introduced vaccination (L . vacce, cow) against small pox, using material from lesions of a similar disease of cattle (cowpox). In 1860s Joseph Lister introduced antiseptic surgery, on the basis of Pasteur’s evidence for the ubiquity of airbone microbes.

Recognition of agenes of infection: first to be recognized were fungi. Agostinod Bassi (1836) demonstrated that a fungus was the cause of disease (of silk worm), the etiologic role of bacteria was established by Koch (1876) for anthrax. The pure culture preparation is the key to the identification. Koch perfected the technique of identification including the use of solid media and the use of stain. After identifying the tubercle bacillus Koch formalized the criteria, introduced by Henle in 1840 but known as Koch’s postulates, for distinguishing a pathogenic from an adventitious microbe:

1. – The organism is regularly found in the lesion of the disease.

2. – It can be isolated in pure culture.

3. – Inoculation of this culture produces a similar disease in experiments on animals.

These criteria have proceeded invaluable in identifying pathogens, but they cannot be met: some organisms such as viruses cannot grow on artificial media and some are pathogenic only for man.

Golden era of microbiology was established between 1860 and 1910 because of development of powerful methodology. Moreover, various members of the German school isolated (in addition to the tubercle bacillus), the Cholera Vibrio, Typhoid Bacillus, Diptheria, Bacillus, Pneumococcus, Staphylococcus, Streptococcus, Meningococcus, Gonococcus and Tetanus bacillus.

Nitrogen Fixation

.    The symbiotic association of cyanobacteria with fungi (lichen), cyanobacteria with bryophytes (Anthoceros), with pteridophytes, (Azolla) with gymnospermes (coralloid root of cycas) and bacteria (Rhizobium, Bradyrhizobiun, zorhizobium, sinorhizobium and mesohizobium etc.) With leguminous plants are under mutual beneficial relationship (symbiosis) in which both the host and bacteria are benefitted. There are number of other angiosperms (excluding legumes) which have symbiotic association with nitrogen fixing microorganisms. About 15 angiospermic plants are of nonlegumes category which fix atmospheric nitrogen, for example, Alnus, Myrica, Purshia, etc. The symbiotic micoorganisms are not only bacteria but also comprises actinomycetes such as Frankia which fixes nitrogen. There are some indications of the existence of haemoglobin like pigment in the root nodules of Alnus, Elaeganus, Shepherdia and Hippophae. In such cases, the hyphal threads of the endophyte fill the cortical cells which increase in volume resulting into a primary nodule recognizable on the root as a ‘swelling’. The lateral roots arise in the vicinity of the primary nodule. Their meristem undergoes branching and gets infected with endophyte results in the formation of a typical adult nodular structure referred to as a ‘rhizothamion’. The occurrence of leaf nodules is confined to the families of Rubiaceae and Myristicaceae. The bacteria is isolated and identified as Mycobacterium rubiacearum, Mycoplana rubra, Flavobacteriumspecies, Phyllobacterium rubicearum and Klebsiella rubiacearum. It is interesting to note that these bacterial isolates do not fix nitrogen.  Several species of Podacarpus possess numerous small nodules on the root system. The most common endophyte is a non-septate fungus resembling the fungal component of endotrophic mycorrhizae. This nodulated root system demonstrates that it performs the process of nitrogen fixation very slowly.

Root Nodulating Symbiotic Bacteria

Rhizobium forms nodules and participate in the symbiotic acquisition of nitrogen. The rod – shaped bacteria, utilize organic acid salts as carbon source without gas formation; while the cellulose and starch are not utilised. The growth is optimum at 27˚C (pH 6.8) and colonies appeared as circular convex semitranslucent, raised and mucilagenous, usually 2-4 mm in diameter. Production of an acid reaction occurs in mineral salt medium. Some strains of rhizobia and agrobacteria show a close relationship in D.N.A. base composition. All species (except Agrobacterium radiobacter ) incite hypertrophies on plant roots. Nodules are incited by strains of rhizobia on roots of leguminous palnts and leaves of certain plants in the families Myristicaceae and Rubiaceae by strains of Phyllobacteria. The strains of Rhizobium are fast – growing where generation time lasts about 6 hours besides showing some other differences with rest of the members of family – Rhizobiaceae.

Some plants bear stem nodules (Sesbania species) by Azorhizobium caulnodans. The strains bear flagella, hence cells are motile (peritrichous flagella on solid medium but one lateral flagellum in liquid medium). They also fix nitrogen. These are oxidase and catalase positive and cannot oxidise mannitol.

The Bradyrhizobium strains are slow growers where generation time about 12 hours or more. The motility occurs by one polar or subpolar flagellum. The growth on carbohydrate medium is accompanied by exopolysaccharide (EPS) slime. Some strains can grow chemolithotrophically (utilizeinorganic salts) in the presence of H2, CO2 and low level of O2. The bacteroids in root nodules are slightly swollen rods with rare branching or occurs forms. Their main symbiotic partner is soyabean, while other bradyrhiozobia produce nodules in the plants such as Lotus, Vigna, Lupinus, Ornithopus, Cicer, Leucaena, Mimosa, Lablab, Acacia and Dalbergia. Now the strains of bradyrhizobia are designated as name of the host plant in parentheses e.g. Bradyrhizobium (Lotus) species.

Recently, it has been observed that some rhizobial strains which are fast growers nodulate soybean, (generally, bradyrhizobia nodulate soybean). These fast growers are identified as a separate genus Sinorhizodium which are rod shaped, usually contain poly-β-hydroxbutyric acid (pH 6-8). Recently several new species have been added.

Most of the rhizobia are discovered only in last decade. Hence, it is not surprising if more and more host which bear bacterial nodules, may not contain the traditional strains of Rhizobium or Bradyrhizobium. Mesorhizobium, a new genus of the family Rhizobiaceae has been named on the basis of whole sequence studies of 16s rRNA. Some of the species of Rhizobium namely, R. loti, R. huakii, R. ciceri, R. mediterraneum and R. tianshanense now known as Mesorhizobium.

Process of Root Nodule Formation

The ‘rhizobia’ live freely in soil and as soon as they come in contact with suitable host, starts the process of infection. There is an initial contact between the bacteria and host which depends upon recognition. Recent evidences suggest that polysaccharides on the surface of invasive bacteria are involved in binding of these cells to constituents (lectins) on the surface of the roots. The factors or proteins located in the nodules are called nodulins while on bacterial surfaces, named as bacteriocidins which help in nodulation. Generally, nodulation starts from the following processes :

(i) Curling and deformation of root hairs : invasion of rhizobia occurs through root hairs. Fine studies of infected root hairs showed the continuation of the wall of the infection thread with the call wall of the root hairs which lend support to the invagination hypothesis. The physiological events leading to infection can be summarised below :

Normal root hair

↓

Exudation of organic substances by roots

↓

Accumulation of ‘rhizobia’ in the rhizosphere

↓

Conversion of tryptophan to IAA

↓

Root hair curling and deformation

(ii) Formation of Infection – thread and Formation of Nodule : it is intresting to note that such binding occurs between compatible (bacteria – host) partners. Tip of curled root hair bends and the bacteria (rhizobial polysaccharides and the DNA) penetrate and grow in the form of an infection tube. Meanwhile, the polysaccharides react with a component of root hair cell to form an ‘organizer ‘. The ‘organizer‘ induces the production of polygalacturonase (PG) followed by depolymerisation of cell wall pectin. In such process, incorporation of rhizobia into cell wall occurs which participate in ‘intussuusception’ i.e. taking in of rhizobia by root hair and its conversion into organic tissues. The infection tube or thread branches into the central portions of the nodule, and the bacteria released into their symbiont’s cytoplasm to multiply. The nucleus of the root hair cell guides the rhizobia.

(iii) Development of nodule : immediately, at the time of release of rhizobia into cytoplasm of the host cortical cells, rapid cell division (called hyperplasia) takes place in the cortical cells. Inside these cells, the bacteria alter their morphology into larger forms called bacteroids. The root cells are stimulated due to this infection to form a tumor like nodule bacteroid-packed cells. The host cells chromosome number of the area become double. The doubling of the chromosome number occurs in nodules of polyploids as well as diploid legumes.

Leghaemoglobin : A red pigment similar to blood haemoglobin is found in the nodules between bacteroids and the membrane envelopes surrounding them. Leghaemoglobin, the prefix ‘leg’ indicates its presence in legume root nodules, is a haemoprotein having a haeme moiety synthesized by the bacteria attached to a peptide chain which represents the globin part of the molecule, is enclosed by a plant gene. The molecular weight of leghaemoglobin is about 16000 – 17000 daltons. The prosthetic group protohaem is synthesized by the bacteroids, while the synthesis of protein part involves the plant cell. The indication of leghaemoglobin enhances the transport of oxygen at a low partial pressure to the nodules and maintains a steady supply of oxygen at low concentration of the nodule. It is not analysed in syanobacterial symbiotic system or in other higher plants such as Frankia and Parasponia which fix nitrogen without leghaemoglobin. The presence of the leghaemoglobin seems to provide full protection against oxygen damage to the nitrogen fixing enzymes.

Bacteria Cause Old Buildings To Feel Off-color

The assumption that time, weather, and pollution are what cause buildings to decline is only partly true. Bacteria are also responsible for the ageing of buildings and monuments – a process known as biodeterioration, where organisms change the properties of materials through their vital activities.

Leonila Laiz from the Institute for Natural Resources and Agrobiology in Seville, Spain, and colleagues have just isolated five new strains of bacteria that degrade old buildings.

Over the last decade, both microbiologists and conservators have been studying the microbial colonization and biodeterioration of both mural paintings in ancient monuments and plaster walls in churches. A specific family of bacteria, Rubrobacter, is commonly found in aged monuments and is thought to be responsible for their rosy discoloration. Until now, only three Rubrobacter species have been identified, and they all thrive in high temperatures of 45 to 80 degrees Celsius (thermophilic bacteria).

Laiz and her team studied three indoor sites showing overt biodeterioration: the Servilia and Postumio tombs in the Roman Necropolis of Carmona in Spain and the Vilar de Frades church in Portugal. Their microbiological and molecular analyses identified five new Rubrobacter strains. The strains are partly involved in the process of efflorescence formation, where salt residues form on buildings, due to the loss of water after exposure to air for a prolonged period of time. Efflorescences lead to damage in the porous structure of the rocks and the gradual deterioration of these buildings.

Two of the newly isolated strains were then grown onto rocks to replicate the biodeterioration process in the laboratory. The Rubrobacter cells penetrated the mineral matrix and crystals formed in contact with the bacterial film. When the film separated from the rock surface after exposure to heat, it removed mineral grains, producing a mechanical deterioration. These three processes are characteristic of biodeterioration and confirm that the isolated bacteria are actively involved in the aging of the studied buildings.

This study of new Rubrobacter that thrive at lower temperatures (non-thermophilic bacteria) gives another insight into the physiology and activity of these bacteria present in monuments.

source – Science daily

Discovery of Agar as a Solidifying Agent

The earliest culture media were liquid, which made the isolation of bacteria to prepare pure cultures extremely difficult. In practice,a mixture of bacteria was diluted successively until only one organism, as an average, was present in a culture vessel. If everything went well, the individual bacterium thus isolateb would reproduce to give a pure culture. This approach was tedious, gave variable results, and was plagued by contamination problems. progress in isolating pathogenic bacteria understandably was slow.
The development of techniques for growing microorganisms on solid media and efficiently obtaining pure cultures was due to the efforts of the German bacteriologist Robert Koch and his associates. In 1881, Koch published an article describing the use of boiled potatoes, sliced with a flame sterilized knife, in culturing bacteria. The surface of a sterils slice of potato was inoculated with bacteria from a needle tip, and then the bacteria were streaked out over the surface so that a few individual cells would be separated from the remainder. The slices were incubated beneath bell jars to prevent airborne contamination, and the isolated cells developed into pure colonies. Unfortunately, many bacteria would not grow well on potato slices.
At about the same time, Frederick Loeffler, an associate of Koch, developed a meat extract peptone medium for cultivating pathogenic bacteria. Koch decided to try solidifying this medium. Koch was an amateur photographer – he was the first to take photomicrographs of bacteria – and was experienced in preparing his own photographic plates from silver salts and gelatin. Precisely the same approach was employed for preparing solid media. He spread a mixture of Loeffler’s medium and gelatin over a glass plate, allowed it to harden, and inoculated the surface in the same way he had inoculated his sliced potatoes. The new solid medium worked well, but it could not be incubated at 37 Degree C because the gelatin would melt. Furthermore, same bacteria digested the gelatin.
About a year later, in 1882, agar was first used as a solidifying agent. It had been discovered by a Japanese innkeeper, Minora Tarazaemon. The story goes that he threw out extra seaweed soup and discovered the next day that it had gelled during the cold winter night. Agar had been used by the East Indies Dutch to make jellies and jams. Fannie Eilshemius Hesse, the New Jersey – born wife of Walther Hesse, one of Koch’s assistants, had learned of agar from a Dutch acquaintance and suggested its use when she heard of the difficulties with gelatin. Agar – solidified medium was an instant success and continues to be essential in all areas of microbiology.

Source – Presscott, Harley, and Klein’s Microbiology.