Sterilisation and Disinfection

Sterilisation is defined as the process by which an article, surface or medium is freed of all living microorganisms either in the begetative or spore state.
Disinfection means the destruction or removal of all pathogenic organisms, or organisms capable of giving rise to infection.
The various agents used in sterilisation can be classified as follows:

A. Physical agents

1. Sunlight
2. Drying
3. Dry heat; flaming, incineration, hot air
4. Moist heat; pasteurisation, boiling, steam under normal pressure, steam under pressure
5. Filtration: candles, asbestos pads, membranes
6. Radiation
7. Ultrasonic and sonic vibrations

B. Chemicals 

1. Sunlight
2. Drying
3. Dry heat; flaming, incineration, hot air
4. Moist heat; pasteurisation, boiling, steam under normal pressure, steam under pressure
5. Filtration: candles, asbestos pads, membranes
6. Radiation
7. Ultrasonic and sonic vibrations

The bacterial cell structure

Bacteria are prokaryotes that lacks well defined nucleuus and membrane bound oranelles like mitochondria and golgiapparatus. These features make them morphologivally and physiologically distinct from eukaryotes.

A prokaryotic cell with three architectural regions :

A cell envelope consisting of a capsule, cell wall and cytoplasmic membrane.

A cytoplasmic region that contains the nuclear material (DNA), ribosomes and various inclusion granules,

Appendages in the form of flagella and fimbria.

Bacterial Cell Structure

 

The structure of bacterial cell organelles and their functions :

Cell envelope : The envelope is a descriptive term for the several layers of material theat envelope the protoplasm of the cell, it is made up of cytoplasmic membrane, cell wall and capsule.

Capsule : Some bacteria have a polysaccharide layer outside the cell wall. This layer is called a capsule. A true capsule is discrete detectable layer of polysaccharides deposited outside the cell wall as seen in Haemophilus influenzae and Streptococus pneumoniae.

A less discrete structure or matrix which embeds the cells would be called a Slime layer

Another type of capsule found in bacteria called glycocalyx,which is thin layer of interwined polysaccharide fibres.

Capsule are generaly composed of polysaccharide; rarely do they contain amino sugars or peptide as in the case of poly d-glutamate capsules in Bacillus anthracis.

Function of capsule :

It protects bacteria from phagocytosis

It protects bacteria from antimicrobial agents.

It also prefers bacterial adherence from many surfaces.

Cell wall : Most prokayotic have rigid cell wall. The cell wall is an essential structure that protects the cell from mechanical damage, osmotic rupture and lysis.

 Cell wall is made up of murein, or peptodoglycan. It is ploymer of disaccharides cross-linked by short chains of amino acids (peptides).

The glycan backbone is made up of alternating molecules of N-ACETYL GLUCOSAMINE (G) and N-ACETYL MURAMICACID (M). This backbone connected to peptide side chain that contains l-alanine, D-glutamate , Diaminopimelic acid, and D-alanine.

Cell wall of Gram Positive & Gram Negative

Ultra Structure of Cell wall of GPC & GNB

Ultra Structure of GPC

Ultra structure of GNB

Chemical structure of Peptidoglycan

The following cell wall characteristics are generally present in a Gram-positive bacterium:

·         cytoplasmic lipid membrane

·         thick peptidoglycan layer

·         teichoic acid and lipids are present, forming lipoteichoic acid which serve to act as chelating agents, and also for certain types of adherence

·         capsule polysaccharides (only in some species)

·         flagellum (only in some species)

·         if present, it contains two rings for support as opposed to four in Gram-negative bacteria because Gram-positive bacteria have only one membrane layer

·         The individual peptidoglycan molecules are cross-linked by pentaglycine chains by a DD-transpeptide enzyme. In gram-negative bacteria, the transpeptidase creates a covalent bond directly between peptidoglycan molecules, with no intervening bridge

Plasma membrane : The plasma membrane is also called cytoplasmic membrane, it is most dynamic structure of prokaryotic cell. It functions as a selective permeability barrieer that regulates the passage of substances into and out of the cell. Since prokaryotic lack the  membrane that plays an ative role in energy generation and biosynthesis.

Structure of plasma membrane : Bacterial membrane are composed of phosholipid and proteins. The arrangement of proteins and lipids to form a membrane is called the fluid mosaic model. Dispersed within the phoshopholipid bi-layer are various structural and enzymatic protiens which carry out most membrane functions.

Functions of prokaryotic plasma membrane :

Osmotic or permeability barrier.

Location of transport systems for nutrients.

Energy generating functions, involving respiratory and photosynthetic electron transport system.

Synthesis of membrane lipids.

Synthesis of cell wall peptidoglycan.

Assembly and secretion of extracytoplasmic protien.

Cytoplasm :  The cytoplasmic consitituents of bacterial cells invaribly include the bacterial chromosome, ribosome and intracellular inclusion granules. Bacteria possess smaller extrachromosomal DNA called plasmid.

Bacterial nuclear material : The total DNA content of a prokayote is referred to as the cell genome. During cell growth and division, the bacterial chromosome is replecated for distribution to progeny cells.

 Bacterial chromosome : It is usually a single molecule of double stranded Dna length of the chromosome is often more than the length of the cell. Hence nucleur material  is believed to be in a super coiled state. Histone-like proteins are complexed with Dna for structural support.

Plasmid : These are extra chromosomal DNA; much smaller in size than chromosome. Plasmids have non-essential genes that would be advantageous for the bacterium under certain conditions. They code for added virulence like toxin production and antibiotic resistance.

Ribosomes : These are structure involved in the process of protein synthesis. The ribosomes of prokaryotes are smaller than that of eukaryotes. Prokaryotic ribosomes are 70S in size, being composed of 30s and 50s subunits.

Mesosomes : They are multiminated structures formed as invaginations of plasma membrane into the cytoplasm. They are functionally analogous to mitochrondria of eukaryotic cells. They are also thought to co-ordinate nuclear and cytoplasmic division during binary fission.

Inclusion granules : cytoplasm of bacterial celll contains different types of inclusions. Inclusions are distinct granules that may occupy a sustantial part of the cytoplasm. Inclusion granules are usually reserve materials. For example, carbon and energy reserves may be stored as glycogen or as poly betahydroxybutyric acid granules.

Volutin granules are reserves of polymeta phosphate and possibly energy. They are also known as the metachromatic granules and are found in Cornybacterium diptheriae.

Flagella : Flagella are filamentous protein structures attached to the cell surface that provide the swimming movement for most motile bacteria. They are composed of protein subunits called flagellin. Bacterial flagella are powered by proton motive force established on the bacterial membrane. About half of the bacilli  and all of the spiral and curved bacteria are motile by means of flagella, very few cocci are motile.

Fimbria : fimbria or pilli are used to designate short, hair-like structure on the surfaces of bacterial cells. They are composed of protein subunits callled pillin. Fimbriae are very common in gram-negative bacteria but occur in some gram positive bacteria as well.

There are two types of pilli; common pilli and sex pilli.

Common pilli : Common pili are usually involved in specific adherence of baceria to surfaces. They are major determinants of bacterial virulance because they allow pathogens to attach to tissue and resist phagocytosis. For example, pathogenic Neisseria gonorrhoeae adhere specifically to the human cervical or urethral epithelium by means of its common pili.

Sex pilli : A  specialized type pf pilus., the F or sex pilus, mediates the transfer of DNA between mating bacteria during the process of conjugation.

Spores : some bacteria have the ability to form highly restant resting stages called endospores. It is formed by a few groups of bacteria as intracellular structures. They are highly restiant to environment sresses such as high temperature, irradiation, strong acids and disinfectants. Endospores are formed by vegetative cells in responce to environment l stress such as exhaustion of an essential nutrient. They germinate and become vegetative cells when the environmentqal stress is relieved. Hence endospore-formation is a mechanism of survial rather than a mechanism of reproduction.

Structure of spores : The spore has a core wall of unique peptidoglycan surrounded by several layers, including the cortex, the spore coat and exosporium. The dehydrated core contains the bacterial chromosomes and a few riboisomes and enzymes.

Why endospores is so helpful in bacterial resistance :

less moisture

less metabolic activity

thick spore coat

presence of calcium dipicolinate; a strong calcium chelator

Introduction to microbial world

Microbiology is the study of microorganisms. It deals with microbial life ; living forms that cannot be prrerciated using naked eye and would require microscopes to magnify them. In 1665 when Robert Hooke observed the surface of cut cork using a light microscope, it was the first view of microscopic life. Later this formed the basis of cell theory.

Microorganisms and their cell structure :

Bacteria are prokaryotic while Fungi and protozoa are eukayotic.

Viruses are also microbes but they are neither prokaryotic nor eukaryotic. Viruses are not considered cellular, they require other cellular machinery for their replication.

Prions are regarded as proteinaceous infectious particles, which lack nucleic acids. They have been linked with diseases like Bovine Spongiform Encephalopathy and Creutzfeldt Jacob Disease.

Beneficial effects of microbiology :

Integral components in our enviornment that aid in nutrient cycling.

Important in food and beverage industries.

Required for antibiotic production.

Best tools in the study of genetic manipulations and hence vital components in biotechnology.

Helpful in the prevention of invasions by pathogenic bactria. Thes role is performed by normal flora; a group of bacteria that live in perfect harmony with human host.

Detrimental effects :

Microbes cause many diaeases in whic some curabele and some are not.

But we should always remember that bacteria which are capable of causing disease represent only a small minority that inhabitat the planet.

Brancehes of Microbiology are :

Bacteriology : Deals with bacteria.

Mycology : deals with the study of fungi.

Parasitology : study of protozoa and parasitic worms (which are not really microscopic)

Virology : Deals with the study of viruses.

Microbial Genetics : Study of how microbes inherit traits like antibiotic resistance.

Nomenclature :

Like other biological names, we use the bionomial system developed by Linnaeus for bacterial nomenclature. According to this every microbe is given two names that is always capitalized and a species name that begins in lower case. The biological name of typhoid bacillus is Salmonella typhi.

Applied areas of Microbiology :

AGRICULTURAL MICROBILOGY

AQUATIC MICROBIOLOGY

INDUSTRIAL MICROBIOLOGY

MEDICAL MICROBIOLOGY

SPACE MICROBIOLOGY

ENVIRONMENTAL MICROBIOLOGY

SOUTHERN BLOTTING

SOUTHERN BLOTTING : E.M SOUTHERN develop southern blotting in 1975. It is used to analyse the related genes in DNA restriction fragements. It also provides the physical map of restriction sites in a particular gene which is located on chromosomes, southern blotting can also gives us information about the number of copies gene in a particular genome,most important it gives us information about degree of similarity of the genes when compared with the other complementry genes.
PROCEDURE OF SOUTHERN BLOTTING :Restriction endonuclease enzyme are used to cut high-molecular-weight DNA strands into smaller fragments.
The DNA fragments of unequal length are then electrophorsed on an agrose gel to separate them by size.
If some of the DNA fragments are larger than 15 kb then prior to blotting, the gel may be treated with an acid, such as dilute HCL, which depurinates the DNA fragments, breaking the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane.
If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydrooxide) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged DNA to a positively charged membrane, single DNA strands are also better for for later hybridization to the probe, and destroys any residual RNA that may still be present in the DNA.
A sheet of nitrocellulose membrane is placed on top of (or below, depending on the direction of the transfer) the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. If transferring by buffer is used to ensure a seal and prevent drying of the gel. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane; ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane.
The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours (standard conditions; nitrocellulose or nylon membrane) or exposed to ultraviolet ray (in case of nylon membrane) to permanently attach the transferred DNA to the membrane.
The membrane is then exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluroscent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA. To ensure the specificity of the binding of the probe to the sample DNA.
After hybridization, excess probe is washed from the membrane, and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.
APPLICATION OF SOUTHERN BLOTSouthern blots performed with restriction enzyme-digested genomic DNA may be used to determine the number of sequences (e.g., gene copies) in a genome. A probe that hybridizes only to a single DNA segment that has not been cut by the restriction enzyme will produce a single band on a Southern blot, whereas multiple bands will likely be observed when the probe hybridizes to several highly similar sequences (e.g., those that may be the result of sequence duplication). Modification of the hybridization conditions (for example, increasing the hybridization temperature or decreasing salt concentration) may be used to increase specificity and decrease hybridization of the probe to sequences that are less than 100% similar.

NORTHERN BLOTTTING

NORTHERN BLOTTTING : It is developed by James Alwine, David kemp, and George stark at standford Uninversity. Nothern blotting. Northern blotting takes its name from its similarity to the first blotting technique, the Southern Blot, named for biologist Edwin Southern The major difference is that RNA, is analyzed in the Northern blot while DNA in Southern Blot. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe. The term 'northern blot' actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane, however the entire process is commonly referred to as northern blotting.
　
PROCEDURE :A general blotting procedure starts with extraction of total RNA from a homogenized tissue sample. The mRNA can then be isolated through the use of oligo (dT) cellulose chromatography to maintain only those RNAs with poly (A)tail RNA samples are then separated by gel electrophoresis by size, are transferred to a nylon membrane through a capillary or vacuum blotting system.
A nylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them. The transfer buffer used for the blotting usually contains formamide because it lowers the annealing temperature of the probe-RNA interaction preventing RNA degradation by high temperatures. Once the RNA has been transferred to the membrane it is immobilized through covalent linkage to the membrane by UV light or heat. After a probe has been labeled, it is hybridized to the RNA on the membrane. Experimental conditions that can affect the efficiency and specificity of hybridization include ionic strength, viscosity, duplex length, mismatched base pairs, and base composition. The membrane is washed to ensure that the probe has bound to only specifically RNA and to avoid background signals from arising. The hybrid signals are then detected by X-ray film and can be quantified by densitometry. 　
APPLICATION OF NOTHERN BLOT :Northern blotting allows one to observe a particular gene's expression pattern between tissues, organs, developmental stages, environmental stress levels, pathogen infection, and over the course of treatment.
The technique has been used to show overexpression of oncogenes and downregulation of tumor-suppressor genes in cancerous cells when compared to 'normal' tissue,
It is also used in gene expression in the rejection of transplanted organs.
If an upregulated gene is observed by an abundance of mRNA on the northern blot the sample can then be sequenced to determine if the gene is known to researchers or if it is a novel finding.
The expression patterns obtained under given conditions can provide insight into the function of that gene.
RNA is first separated by size, if only one probe type is used variance in the level of each band on the membrane can provide insight into the size of the product, suggesting alternative splice products of the same gene or repetitive sequence motifs. The variance in size of a gene product can also indicate deletions or errors in transcript processing, by altering the probe target used along the known sequence it is possible to determine which region of the RNA is missing.

HISTORICAL INTRODUCTON TO MEDICAL MICROBIOLOGY

Medical microbiology deals with the study of microoganisms that cause disease in human beings. earlier it was belived that living oranisms could arise from non-living matter. This view that disease was spontaneously generated form non living matter. This view that disease was sponaneously generated instead of being created by microorganisms which grow by reproduction, was popularly called the Theory of spontaneous Generation. It was disproved by Pasteurs's experiments using swan neck flask. His results provided definitive results to support the theory of Biogenesis- idea that living cells arise from pre-existing living cells.
After that the golden era started of Microbiology.

ANTONY VAN LEEUWENHOECK - FATHER OF MICROSCOPY : It was he who first described bacteria, free-living and parasitic microscopic protists, sperm cells, blood cells microscopic nematodes.

LOUIS PASTEUR : FATHER OF MICROBIOLOGY : He began his carrer as a chemist. He described the scientific basis of fermentation and wine making. Pateurs intuited that if germs were the cause of fermentation, then it could be also cause of contagious diseases. This proved to be true for many diseases such as potato blight, silkworm diseases, and anthrax. Pasteur's work gave birth to most important theoractical concepts and practical application of modern science. The term vaccination is given by him. He solved the mysteries of rabies ,anthrax, chicken cholera and silk worm diseases, and contributed to the first development of vaccines.

ROBERT KOCH AND POSTULATES :
The microbe must be present in every case of the disease.
The microbe must be isolated from the diseased "host" and gorwn in pure culture.
The disease must be reproduced when the pure culture is introduced to a non disease susceptible host.
The microbe must be recorverable from an experimenantly infected host.
In 1882 he discovered the mycobacterium tuberculosis as the cause of tuberculosis. He went on to lead an expendition in 1883 to india and egypt and discovered the causative agent of cholera - Vibrio cholera. Koch became the first to grow bacteria in colonies, first on potato slices and later with his pupil "petri" on solid gelatin media. He was the first to use agar to solidify culture media.

ELIE METCHNIKOFF AND PHAGOCYTOSIS : He was russian zoologist, much facinated by the work of pasteur, he wrote to him asking for postiion in his laboratries ; in a short time he became one of the principal collaboratiors with pasteur, especially in works concerning immunity. He was intrigued by the motile cells of transparent starfish larvae and observed that few hours after introducing a rose thron into the larvae, he extended his stud yo mammalian leukocytes that led to the famous discovery of phagocytosis, he was awarded with nobel prize in 1908.

EDWARD JENNER- FATHER OF MODERN VACCINATION : In 1980 as a result of janners discovery , The world health organization officially declared "the world and its people " free from small pox.

JOSEPH LISTER : FATHER OF ANTISEPTIC SURGERY : In earlier time the survival rate was very low after the surgery becauase of infection. Lister covered the wound with lint carbolic acid so that the wound will not welcome the microorganism. He then devised a machine that pumped out a fine mist of carbolic acid into the air around during an operation. So basically he brings the concepts of sterillization of operation theatre.