Elzu's micro notes

  Pulsed Electric Fields   Pulsed electric fields PEF is a non-thermal method of food preservation that uses short pulses of electricity for microbial inactivation and causes minimal damaging effect on food qualities. PEF technology aims to offer consumers high-quality foods. High Intensity Pulsed Electric Field (PEF) Processing involves the application of pulses of high voltage (typically 20-80 kV/cm) to foods placed between 2 electrodes . PEF may be applied in the form of exponentially decaying, square wave, bipolar, or oscillatory pulses and at ambient, sub-ambient, or slightly above ambient temperature for less than 1s. Energy loss due to heating of foods is minimized, reducing the detrimental changes of the sensory and physical properties of foods. PEF treatments showed effects on the microbial inactivation in milk, milk products, egg products, juice and other liquid foods Some important aspects in pulsed electric field technology are the generation of high electric f

The three basic stages of protein synthesis are initiation, elongation, termination. These process is almost similar in prokaryotes and eukaryotes. Initiation in prokaryotes This involves mRNA molecule, a ribosome, a specific initiator tRNA, Initiation factors(proteins) GTP and magnesium ions. Steps involved: ·         Binding of 30Ssmall ribosomal unit to the region of the mRNA with AUG initiation codon. Actually it binds to a sequence upstream of AUG which is known as Ribosome binding site(RBS). Most of RBS are 8 to 12 nucleotides upstream from initiation codon.   This is a purine rich region almost similar to AGGAG and complementary to a pyrimidine rich region (UCCUCC) at 3’ end of 16SrRNA. RBS   was evident from work of John Shine and Lynn Dalgarno.   So this region is also known as Shine- Dalgarno sequence . Mutation occur to Shine- Dalgarno sequence or sequence complementary to it, mRNA translation will be abolished. ·          The ribosomal subunit comes to the mRNA

Characteristics of Genetic code Triplet code A single base cannot be codes because there are 20aminoacids and only 4 bases. Pairs of bases also cannot serve   as codons because there are only   4 2 = 16 possible pairs   of 4 bases .Triplets of bases are possible because there are 4 3   =   64. triplets which is more than adequate . Ø   Triplet code is minimum required to code for 20 amino acid . There are certain trends for patters of genetic codes . Ø   Aspartic acid codons are similar ( GAU , GAC ) to   glutamic acid codons(GAA,GAG) the difference only exhibited in the third base . Ø   Codes for aromatic acids phenylalanine ( UUU , UU C ) and tyrosine (UAU,UAC) and tryptophan UGG    all begins with uracil First two bases are assigned to 5 amino acids GC –Alanine GG- Glycine CC-Proline AC-Threonine GU- Valine All codons with A in the second position specify the changed amino acid except arginine. All the acidic and basic amino acids have A or G as the sec

GENITIC CODE How do nucleotides in mRNA  molecules specify the AA the segment in proteins ? With 4 different nucleoids ( A , C G , U ) a three letter code generate 64 possible codes. Genetic code : collections of basic sequences that respond to each  amino acid and stop signals  for translate . 1960- Francis Crick , Leslie Barnett , Sidney Berner , Watts Tobin There are several ways in which a codes could be read from a mRNA  molecule . Two most important ways are : Ø   Overlapping codes Ø   Non overlapping codes   In overlapping model Each base serves as  first base of some codon . After the first amino acid  in a protein is coded the next two and for that matters the remaining amino acids in the protein are particularly pre determined . Non overlapping model Mutation allow change in one base single mutagenic base change could after as many as 3 amino acids .  If mRNA  strand      :  ABC  DAB CDA In Overlapping model ABC       BCD     CDA   

Wobble hypothesis This hypothesis was proposed by Francis Crick in 1965 According to this hypothesis, the complete set of 61 sense codons can be read by fewer than 61 distinct tRNAs because pairing properties of the bases in the anticodon. Specifically , the base at the 5’end of the anticodon complementary to the 3’ end of the codon is not constrained three dimensionally as the other 2 bases. This feature allows for less exact base pairing ,so that the base at 5’end of the anticodon can pair with more than one type of base at the 3’end of the codon or it can wobble. No single tRNA molecule can recognize 4 different codons .But if the tRNA molecule contains the modified purine inosine(I) at the 5’end of the anticodon ,then that tRNA can recognize 3 different codons (A,U,C) G Nucleoside at 5’ end of anticodon    can pair with U or C C Nucleoside at 5’ end of anticodon    can pair with G A Nucleoside at 5’ end of anticodon    can pair with U U   Nucleoside at 5’ end of antic

Protein synthesis take place on ribosomes, where the genetic message encoded in mRNA is translated .The mRNA is translated in the 5’ to3’ direction and the polypeptide is made in the N terminal to C terminal direction. Aminoacids are bought to the ribosome bound to tRNA   molecules. The correct amino acid sequence is achieved because of The binding of each   aminoacid to its own specific tRNA The binding between the codon of the mRNA and the complementary anticodon in the tRNA mRNA recognizes tRNA codon was proved by G Von Ehrenstein, B Weisblum and S Benzer.T hey concluded that specificity of codon recognition lies in the tRNA molecule not in the amino acid it carries. Adding an amino acid to tRNA The correct amino acid is attached to the tRNA by enzyme aminoacyl –tRNA synthetase.The process is called aminoacylation or charging and it produces aminoacyl tNA or charged tRNA. Amino acylation uses energy from ATP hydrolysis. Since there are20 different amino acids, there ar

Aim To determine the antibiotic sensitivity of the given organism by Kirby – Bauer disc diffusion method. Principle Kirby – Bauer disc diffusion method is commonly employed for antibiotic sensitivity test. The test is based on the fact that for a given antibiotic, the size of zone of inhibition is related to MIC                  {Minimum   Inhibitory Concentration }. MIC is referred as the lowest concentration of the antibiotic that exhibits the zone of inhibition on the assay plate. Kirby – Bauer Agar disk diffusion method provides qualitative interpretive category results of susceptible, intermediate, and resistant bacterial isolate Materials required ·          Sterile forceps ·          Sterile swabs ·          Broth culture of the organism ·          Sterile Mueller-Hinton agar plates ·          Antibiotic discs   Procedure Preparation of plates Sterile Mueller-Hinton agar [pH 7.3] was prepared and poured into plates [the depth of the medium should be a

Cl.botulinum Ø   Causes   botulism( Sausage poisoning/fatty poisoning) Ø   A paralytic disease mainly through food poisoning MORPHOLOGY ·         Gm+   ve rod shape bacteria ·         Non capsulated ·         Motile   by peritrichous flagella ·         Produce spores CULTURAL CHARACTERS §   Strict anaerobe §   Optimum temperature   for growth is 35 o C §   Shows good growth in ordinary media §   Sporulate better in alkaline glucose   gelatin media at   20-25 C . RESISTANCE ·         Spores are   heat     resistant    and      radiation   resistant    .  CLASSIFICATION ·         Cl.botulinum are classified into 8 types on   basis   of immunological difference in toxins . ·         C2   -cytotoxin ·         Others   produce   neurotoxin , powerful exotoxin. FEATURES OF EXOTOXIN ·         It is not released during life time of organism and appears in medium only the   death or autolysis of cell. ·         It is produced intracellularly ·     

Hyperchromic shift on DNA melting Aim To   demonstrate   hyperchromic   shift   on   DNA   melting. Principle Hyperchromicity is the an   increase   in the absorbtion of ultraviolet light by a solution of DNA when subjected to heat, alkaline conditions, etc. Hyperchromicity of DNA occurs when the DNA duplex is denatured .The UV absorbance   is increased when the DNA strands are being separated, either by heat or by addition of   denaturant or by increasing   pH level. Heat denaturation of DNA is also called melting when DNA in solution is heated above its melting temperature [usually more than 80 o C ], the double stranded DNA unwinds to form single stranded DNA. The bases becomes unstacked and can thus absorb more light. The G + C content often determined from the melting temperature [Tm] of DNA . In double stranded DNA ,three hydrogen bonds joins G C base pairs, and 2 hydrogen bonds connect AT bases pairs. DNA with a greater G + C content have more hydrogen bonds and it

Genus leptospira consist both pathogenic and saprophytic species. L. interrogans are medically important species. They are again classified into many serotypes.  Morphological features 6-20 µm length 0.1 µm thickness They are actively motile, Gram negative bacteria ,mainly stained by special stains Giemsa stain and silver impregnation method. They can also seen under dark ground illumination and electron microscopy. Cultural characters Enriched media with rabbit serum, Korthof’s media, Stuarts media, Fletcher’s media are used for cultivation. EMJH media is commonly used. Optimum temperature for incubation is 25-30 o C with a pH of media 7.2-7.5 . Leptospira grows in aerobic or microaerophilic conditions. It can be inoculated into guinea pigs     and also could grow in chorioallantoic membrane of chick embryos. Resistance Leptospira is susceptible to heat ,sensitive to bile acids and disinfectants. Pathogenicity Rats are the natural reservoir, infection is usually as

ESR determination by Westergren’s method Aim To determine the erythrocyte sedimentation rate of the given sample. Principle The erythrocyte sedimentation test is a nonspecific test. It is raised in a wide range of infections, inflammatory, degenerative and malignant conditions associated with changes in plasma proteins particularly increases with fibrinogen, immunoglobulins and C reactive proteins. The ESR is also affected   by many other factors including anaemia, pregnancy, haemoglobinopathies and treatment with anti-inflammatory drugs. Moderately raised sedimentation rates can sometimes be found in healthy people, particularly those living in tropical countries and a normal ESR cannot exclude disease. When performed, test results must be interrupted in conjugation with clinical findings and the results of other laboratory tests. When anticoagulated   blood in a vertically positioned Westergren’s pipette is left undisturbed, red cells aggregate ,stack together to form rou

Helicobacter     pylori Helicobacter pylori  is a spiral bacteria discovered by  two Australian scientists Warren and Marshall in 1983. Today H pylori  colonies the stomach of  half the human population of the world. Usually it infects from early childhood . Helicobacter pylori   is adapted to human gastric mucosa. The presence of helicobacter was detected  even in the intestine of pre Columbian mummies in USA. Helicobacter pylori  was originally named as Campylobacter pylori   but as there was differences from Campylobacter it was renamed as Helicobacter pylori. Morphological characters Helicobacter pylori  is a gram negative spiral  bacteria , motile by lophotrichous  flagella. In old culture it appears  in coccoid form. Biochemical characters It produces oxidase , catalase , phosphatase and hydrogen sulphide. A distinctive property of this bacteria is abundant production of urease. This property is used as a rapid diagnostic tool with gastric biopsy samples. It does not ferment carb