Coupon Accepted Successfully!


Important Points

  • Study of genes, their structure, function, heredity and variation
  • Bacteria obey the laws of genetics
  • Genetic information contained in DNA
  • Central dogma of molecular biology


  1. Structure of DNA molecule
    1. Double helix
    2. Pyrimidine bases (Thymine, cytosine) AND Purine bases (Guanine, adenine)
    3. Pair A=T; G=C
    4. Dexoyribose sugar
  2. Structure of RNA
    1. Ribose sugar and Uracil instead of Thymine
    2. rRNA: Synthesis of proteins
    3. tRNA : Accepts single amino acid and transfers it to a ribosome
    4. mRNA: Template for translation

A. Special point

  1. Genetic information stored as code: codon
  2. Triplet of bases
  3. Each code specifies for a single amino acid and more than one code may exist for a single amino acid
  4. Code is therefore said to be degenerate
  5. Start codon: AUG
  6. Nonsense codons: UAA, UAG, UGA
  7. Shine dalgarno sequence: present upstream of start codon, required correct alignment on mRNA in within the two subunits of ribosome
  8. Cistron/gene: Segment of DNA carrying a number of codons specifying for a particular polypeptide is known as cistron/ gene
  9. Locus: large number of genes
  10. Genome: all the loci

B. Extra chromosomal genetic elements

  1. Plasmids: Extra chromosomal genetic elements that can replicate autonomously and can maintain in the cytoplasm of a bacterium for many generations.
  2. Circular piece of DNA. Not essential for normal life, confer on it additional properties: drug resistance, bacteriocin production, toxigenicity etc.
  3. Information for controlling its replication
  4. May get integrated into host genome: Episome
  5. Some confer ability to conjugate (F Plasmid): Conjugative/ self transmissible plasmid 
C.  Plasmid determined properties
  1. Resistance to:
    1. Antibiotics
    2. Heavy metal ions 
  2. Metabolic functions
    1. Citrate utilization
    2. Fermentation of lactose, raffinose, sucrose 
  3. Virulence factors
    1. Entero toxins
    2. Colicin
    3. Exfoliative toxin etc.

D. Genotypic/ phenotypic variations

  1. Phenotype: Characteristics expressed by a cell in a given environment
  2. Genotype: Collection of genes encoding these characteristics
  3. Bacteria are very adaptable, may alter their phenotype in response to environmental change, while genotype remains unchanged.
  4. Not all genes of bacteria express all the time
    1. Typhoid bacillus: No Flagella when growth in phenol agar
    2. Synthesis of β galactosidase: Produced only when lactose is present in the medium (induced enzymes)
    3. Economy of nature


  1. Random, undirected, heritable variation caused by an alteration in the nucleotide sequence
  2. Addition/deletion/ substitution of one/more bases
  3. One per 102-1010 cell divisions
  4. Mutants will outnumber and overgrow if their new character makes them better fitted to grow under the prevailing conditions: Selection of mutants
  5. Frequency increased: Nitrogen mustard, acriflavine, mitomycin C, 5- bromouracil, 2- aminopurine
  6. Missense mutation: Triplet code altered, codes for amino acid different from that normally located at a particular position
  7. Nonsense mutation: Premature polypeptide chain termination
  8. Transversion: Substitution of purine for pyrimidine and vice versa
  9. Lethal mutation: Involve vital functions, conditional lethal mutants: Live under permissive conditions but not under non permissive conditions
  10. t.s. (Temperature-sensitive mutant) : Live at 35°C; Dies at 39°C
  11. Most mutations unrecognized
  12. Test to determine whether a chemical is mutagen or carcinogen: Ames test

A.  Acquisition of new genes

  1. Mutation
  2. Acquiring new DNA from external source
    1. Transformation
    2. Transudation
    3. Lysogenic conversion
    4. Conjugation 

B.  Transformation

  1. Acquisition of DNA from environment and incorporation in its genome
  2. First demonstrated in S. pneumoniae. Subsequently also demonstrated in other bacteria like B.  
  3. Subtilis, H. influenzae, N. gonorrhoeae
  4. DNA must be derived from closely related strain
  5. Cells that can take up naked DNA are called as competent
  6. First demonstrated by Griffith 1928
  7. Transformation can be inhibited by DNAases 

C.  Transduction

  1. Transfer of a portion of DNA from one bacterium to another by bacteriophage
  2. Generalized transduction:
  3. Due to phages that lyse host cell: Virulent phages
  4. During assembly, phage head is filled with host cell DNA (packaging error)
  5. Such a phage infects a second bacterium, DNA enters
  6. Transduce any gene
  7. Size similar to phage genome
  8. B/w closely related strains
  9. Chromosome DNA as well as plasmid DNA e.g. penicillin resistance in S. aureus  

D. Specific transduction:

  1. Due to phages incorporated into host genome: Temperate phages (Lysogenic)
  2. When infected with temperate phage: small proportion get incorporated into host cell genome: In some lysogenic cell lytic cycle is resumed The prophage (integrated phage DNA) is excised In some cells prophage is excised inaccurately so that neighboring portion of bacterial DNA is also removed Such piece of DNA is transduced into second cell and integrates to the specific site.

E.  Lysogenic conversion:

  1. In lysogenic bacteria, prophage codes for new characters
  2. Corynebacterium diphtheriae; diphtheria toxin is coded by β phage
  3. S. pyogenes; Dick toxin is coded by a bacteriophage 

F.  Conjugation

  1. Transfer of DNA that occurs during contact b/w bacterial cells
  2. Common among gram negative bacteria
  3. Plasmid - codes for Sex Pilus, 1-2 μm length
  4. Pilus attaches to the surface of recipient cell and holds the two cells together
  5. The plasmid DNA replicates and a copy of it passes through the sex pilus
  6. F-Factor: genetic information for synthesis of sex pilus, which is required for self transfer
  7. F-Factor has an ability to integrate in the host chromosome: Hfr cells
  8. F-Factor with host chromosomal genes: F' Factor (transferred by sexduction)
  9. R- Plasmids: RTF + r, Resistance to eight or more drugs may be transferred simultaneously,
  10. most common method of resistance transfer.
  11. E. coli, K. pneumoniae, Salmonella, Shigella, Pseudomonas 

G. Transposable genetic elements

  1. Transposition requires no homology between transposable elements and its site of insertion
  2. 'Jumping Genes’: Move from one plasmid to another, to a phage or to the bacterial chromosome
  3. Do not contain genetic information necessary for their own replication
  4. IS (insertion sequences): small, cryptic (1-2 kb)
  5. Transposon: Large (4-25 kb) encode at least one function, has a segment of DNA coding for one/more genes at the centre and two ends carrying inverted repeat sequences. Single stranded loop and double stranded stem. 

H. Recombination dna technology

  1. Insertion of foreign DNA molecule into DNA of a vector, which can replicate autonomously in a
  2. suitable host
  3. DNA endonucleases/ restriction endonucleases: Cut DNA at specific sequences
  4. Produced as part of defense against the insertion of foreign DNA
  5. Target site comprise of four- six base pairs which are 'Palindromic'
  6. Blunt ends; Sticky ends
  7. Rejoined with ligase
  8. Uses:
    1. Vaccine production: Hepatitis B Vaccine
    2. Antigen production: gp 120 of HIV
    3. Proteins: Human growth hormones, insulin, interferons
    4. Gene therapy 

I.   Nucleic acid probes

  1. Single stranded nucleic acid that can hybridize specifically with its complementary strand
  2. Detect RNA/DNA
  3. Components:
    1. The probes
    2. The target
    3. Reporter molecule
      1. Culture confirmation: M. Tuberculosis, M. kansasii, MAC, M. gordonae
      2. Detection of fastidious organisms: N. gonorrhoeae, C. trachomatis

J.   Polymerase chain reaction

  1. Discovered by Kary Mullis
  2. Primer mediated temperature dependent technique for enzymatic amplification of a target sequence to such an extent that it can be detected.
  3. Target DNA
    1. Primers
    2. Taq Polymerase
    3. Nucleotides
  4. PCR Steps

5. Detection of amplified sequence by
  1. Gel electrophoresis
  2. Southern blotting
  3. Colorimetric method

Extra Edge :-

  1. Post antibiotic effect-
    influence of antibiotic challenge can persist even after the drug has been excreted or has been degraded
    Nucleic acid based technologies for detection of microbial infections
  2. Nucleic acid amplification techniques
    1. In vivo- culture enrichment
    2. In vitro
      1. PCR - Polymerase chain reaction
      2. LCR - Ligase chain reaction
      3. NASBA - Nucleic acid sequence-based amplification
      4. SDA - Strand based amplification
      5. QBRDA Q -beta replication-dependent amplification
  3. Amplified target detection techniques
    1. Agarose gel electrophoresis
    2. Dot or Slot blot hybridization
    3. Southern procedure 

Direct target nucleic acid detection techniques

  1. In-situ hybridization
  2. Restriction fragment length polymorphism analysis (RFLP)
  3. Pulse field gel electrophoresis (PFGE)

Recent Advances :

  1. Integrins- site specific recombination systems that mediate the movement of small DNA elements called gene cassettes
  2. DNA Microarrays monitors whole genome on a miniaturized glass substrate or “chip” enabling visualization of interactions among thousands of genes simultaneously & has been applied to pathogen identification & classification, polymorphism detection & detection of drug resistance mutations for viruses & bacteria
  3. Quorum sensing – the term is used to describe the phenomenon whereby accumulation of a diffusible low molecular weight signal molecule enables individual bacterial cells to sense when the minimum number , or ‘quorum’, of bacteria has been achieved for a concerted response to be initiated.

K.  Sterilization and disinfection

  1. Sterilization: Process by which an article, surface, medium is freed of all microorganisms including viruses, bacteria, their spores & fungi
  2. Disinfection: Process of destruction of organisms capable of giving rise to infection: (bacterial spores not killed)
  3. Antisepsis: Destruction of microorganisms in living tissues. A disinfectant applied to living tissue is referred to as an antiseptic
a.  Physical Agents b. Chemical Agents
Sunlight Phenols/ cresols
Drying Halogens
Heat Metallic salts
Filtration Aldehydes
Radiation Alcohol
  Vapour-phase disinfectants
  Surface active disinfectants

A. Physical Agents

1.  Heat

a. Dry heat Kills by oxidation
b. Red heat
: indicating wires, loops, points of forceps
c. Flaming:
Scalpel blades, glass slides, mouth of culture tubes
d. Hot Air:
160°c for 1hr

  1. Sterilization of glassware: Glass syringe, test tube, petri dishes, pipettes, flasks
  2. Metal instruments: Forceps, scissors, scalpels
  3. Oils, jellies, powders
  4. Controls: Biological: Bacillus subtilis subspecies niger 106 spores (filter paper strip)
  5. Chemical control: Browns tubes (redgreen)

e.  Moist heat:

  1. Kills by denaturation and coagulation of proteins
  2. Temperature below 100°c
  3. Temperature at 100°C :  boiling water; free steam
  4. Temperature above 100°c

f.   Temperature below 100°c

  1. Heat labile fluids disinfected
  2. Pasteurization - 63°C 30 min (Holder)
    72°C 20 sec (Flash)
  3. Serum bath 56°C , 1hr
  4. Vaccine bath 60°C, 1hr
  5. LTSF (Low Temperature Steam- Formaldehyde sterilization)- Method of sterilization
  6. Steam at subatmospheric pressure at Temp: 75°C

g.  Moist heat, temp at 100°c

  1. Boiling at 100°C: 10-30 minutes, Some bacterial spores not killed, Metal, glass, rubber items
h.  Free steam at 100°c
i. Latent heat
ii. Culture media containing sugar, gelatin

iii. Some spores not killed

iv. Tyndallization/ intermittent sterilization: 100
°c, 20 minutes for three consecutive days
i.    Moist heat, temp >100°c
i. Steam under pressure (autoclaving)
ii. 15 psi, 121°c, 15-20 min
iii. Dressing material, linen, gloves
iv. Culture media, aqueous solution
v. Controls: Bacillus stearothermophilus 106 spores
vi. Chemical: brownes tube no 1

2.  Filtration

  1. Liquids such as sera, solutions of heat labile substances - sugars, urea sterilized by filtration
  2. Mycoplasma, viruses cannot be kept back by the bacterial filters
  3. Controls: Serratia marcescens, Pseudomonas diminuta
  4. Earthenware filters
    i.    (Kieselguhr) fossil diatomaceous earth
    ii.    Chamberland made of unglazed porcelain
  5. Asbestos (seitz) filters
     i.    Disc of magnesium trisilicate
  6. Sintered glass filters-reusable discs
  7. Membrane filters : cellulose esters (0.015- 12m), most commonly used filters. Syringe filters
  8. Air filters: HEPA remove particles more than 0.3m 

Extra Edge :

  1. Distill water should have conductivity < than given by 1ppm NaCl
  2. Deionized water (demineralized water) should be equivalent to double
  3. Glass-distilled water and should have very low conductivity

 Recent Advances

  1. In Ion exchange resins used to deionize water:
    a. Cation exchanger column is regenerated by 10% aqueous HCl
    b. Anion       ,,                      ,,                   ,,     10% aqueous NaOH 
  2. McFarland standards – are opacity standards used to prepare a bacterial suspension of a standard concentration eg 0.5 McFarland standard opacity corresponds to bacterial concentration of 150 millions/ml = 100 x 1000,000
  3. Gnotobiotic animals- are ‘germ free’ animals reared in sterile isolator are abnormal animals ( structurally & physiologically) but are valuable in some kinds of microbiological experiments
  4. Hydroclave is more sophisticated method of autoclaving in which there is no direct contact of steam with waste. Moisture content of waste turns into steam which builds required pressure in the chamber
  5. HEPA filters in biological safety cabinets are sterilized by hydrogen peroxide
  6. Gas plasma sterilization uses hydrogen peroxide as substrate gas & radiofrequency emissions to generate plasma at low temperature. Used for sterilization of heat sensitive materials, instruments & for sterilization against prions.
  7. Chemical disinfectant for biological safety cabinets is 40% formaldehyde
  8. Sodium dichloroisocyanurate (NADCC)- solid chlorine releasing granules for direct application to spillages
  9. NEW methods/agents of sterilization
    1. Surfacine - Antimicrobial coating containing silver iodide
    2. Sterilox - Superoxidized water
    3. Endoclens - Liquid sterilization system for endoscopes

Attest   - Rapid read out ethylene Oxide biological indicator producing fluorescent change on failure   

3.  Radiation

  1. Non-ionizing : UV radiation 250-260 nm, spores resistant, HIV not inactivated. Induce thymidine/ pyrimidine dimers, disinfection of clean surfaces.
  2. Ionizing: α rays, gamma rays, cosmic rays, sterilization of pre packed disposable items (cold sterilization) Control: Bacillus pumilis

b. Chemical Agents

1.  Phenols and Cresols

  1. Cause cell membrane damage
  2. Resistant to inactivation by organic matter
  3. Active against Gram +, Gram -, moderately active against Mycobacteria
  4. Little activity against spores
  5. Use: Discarded cultures, pipettes, other infected material
  6. Phenol: bactericidal 3-5% 

Recent Advances :

NIOSH approved N95 disposable particulate respirators (Filtering face pieces/masks) are designed to help provide respiratory protection for the wearer. They have filter efficiency level of 95% or greater against particulate aerosols free of oil when tested against a 0.3 micron particle. The “N” means “Not resistant to oil”. The “95” refers to a 95% filter efficiency


2.  Halogens

  1. Chlorine, hypochlorites, inorganic/organic chloramines
  2. Bactericidal, sporicidal, virucidal, little activity against M. tuberculosis
  3. Release of free chlorine strong oxidizing agent
  4. Activity decreased by presence of organic matter
  5. Iodine:
  6. Alcoholic/aqueous solutions: skin antiseptic
  7. Also active against M. tuberculosis
  8. Iodophores: Mixture of iodine with surface active agents, best antiseptic, eg. Betadine

Recent Advances):-

  1. BioFilm are formed by Coagulase negative staphylococcus, staphylococcus aureus, Enterococcus, K. pneumoniae Pseudomonas aeruginosa. Dental Plaque is biofilm known to cause disease.
  2. Bacteriocins are a group of highly specific antibiotic like proteinaceous substances produced by certain strains of bacteria that kill other strains of the same or different species eg.
Colicins & Micronics Enterobacteriaceae
Klebocins K. pneumoniae
Pyocins Pseudomonas aeruginosa
Aureocin S. aureus A70
3.  Metallic salts
  1. Mercury: combines with -SH group of bacterial proteins e.g. merthiolate: preservation of sera
  2. 1% silver nitrate: prophylaxis for gonococcal ophthalmia 

4.  Aldehydes

  1. Formaldehyde
    1. Irritant, water soluble gas; Lethal to bacteria, spores, fungi, viruses
    2. Cheap
    3. Sterilization of rooms, furniture, clothing blankets, mattresses
    4. Less effective in presence of organic matter 
  2. Glutaraldehyde
    1. More effective, less irritant
    2. 2% solution(Cidex): sterilization of heat sensitive instruments cystoscopes, bronchoscopes (Fiber    optic scopes) for at least 3 hrs at alkaline pH       

5.  Alcohols

  1. Kill Bacteria, no action on spores, viruses
  2. 60-70%; presence of water essential
  3. Isopropyl alcohol: Better fat solvent, more bactericidal, less volatile

6.  Vapour phase disinfectants:

a.  Ethylene oxide (e.o.)

                                i.    Mechanism of Action : Alkylation Reaction especially of nucleic acids


                               ii.    Various types used       

  • Pure E.O.: Highly explosive and flammable used hence can he in self contained system only
  • E.O. + Chlorofluoro Carbon:
  • CFC causes depletion of ozone layer.  Banned Now
  • E.O.+CO2
  1. High pressure mixture
  2. Change in composition with time leading to increase in E.O. conc.
  3. Acid formation - corrosion

           iii.    ETO Sterilization depends upon:

  • Concentration of E.O. : usually > 700mg/l
  • Temperature   : 2 types of cycles
  1. Cool cycle : runs at 37°C
  2. Hot cycle : 47 — 66°C (Average 55)

           iv.    Biological Control : Spores of Bacillus globigi


b.  Side Effects :

i. Acute Reactions

  • Headache, Nausea, Vomiting, Dizziness
  • Bums, Blisters
  • Severe toxic shock in cardiopulmonary bypass surgery patients
  • Hypersensitivity reactions in haemodialysis patients

          ii.    Chronic Reactions

  • Mutagenic
  • Peripheral neuropathy 
c.  Precautions during use
  1. Installation : Away from general area
  2. Kept at Negative pressure
  3. Minimum of 10 air exchange / hour in area having ETO
  4. Sterilizer
  • Immediate exhaust on opening the door of sterilizer
  • Aeration of Load is must

           v.    PEL (Permissible Exposure Level of ETO) is - 1PPM / 8 hours


           vi.    STEL (Short time exposure limit) 5PPM     


d.  Hydrogen peroxide –

  1. Broad spectrum antimicrobial activity with confirmed virucidal, bactericidal, fungicidal, myco bactericidal, and sporicidal activity.
  2. In the gas phase, hydrogen peroxide (also known as VHP for Vaporized Hydrogen Peroxide) demonstrates significantly greater antimicrobial efficacy than in the liquid form.
  3. A type of low temperature sterilization.
  4. Some systems only use hydrogen peroxide gas under vacuum
  5. Some use hydrogen peroxide in the presence of plasma.
  6. A plasma is essentially excited H2O2 produced by adding energy (in the form of heat or an electromagnetic field).
  7. The systems have a rapid cycle time (<1 hour)
  8. Provide sterile instruments for immediate use or sterile storage (with no extended aeration requirements as traditionally needed with ethylene oxide and formaldehyde)

7.  Surface active disinfectants- MOA: Reduction of surface tension

  1. Possess both hydrophobic and hydrophilic groups
  2. Anionic, cationic, non-ionic, amphoteric   
  3. Cationic, quaternary ammonium compounds, most important
  4. Bacteriostatic
  5. Weak detergent
  6. Gram +> gram -ve
  7. Active against enveloped viruses
  8. Anionic, common soap
  9. Strong detergent
  10. Weak antimicrobial
  11. Amphoteric, tego compounds
  12. Detergent properties of anionic compounds
  13. Antimicrobial activity of cationic compounds
Extra edge:

Low temp sterilization

  1. ETO             
  2. H2O2   
  3. Low temp steam form-aldehyde


Testing the efficacy of disinfectants

  1. Minimum inhibitory concentration (MIC): Lowest concentration of the disinfectant that inhibits the   growth of Salmonella typhi
  2. Phenol coefficient test
  3. Similar quantities of organisms added to rising dilutions of phenol and the disinfectant to be tested
    i.    Rideal-Walker: No organic matter added
    ii.    Chick Martin: organic matter added
    iii.    Dilution of disinfectant which kills the organisms divided by dilution of phenol
     iv.    Phenol coefficient      1.0 Equal
    <1.0 Less                >1.0 More
  4. Capacity test (Kelsey and Sykes test)
  5. In use tests







Recent Advances

  1. SOP’s- ‘Standard operating procedures’ is a written set of instructions for every test process or procedure, to set minimum acceptable standard of a laboratory. Also called laboratory procedure manual or bench book.
  2. QA = QC + IQA + EQA  (QA: quality assurance; QC: quality control; EQA: external quality assurance)
  3. QC is applied to analytical phase of testing process

Classification of pathogens into Hazard groups

   Hazard        Group


Containment level


Organism most unlikely to cause human disease



May cause human disease,

May be hazard to laboratory workers

Unlikely to spread in community,

Effective prophylaxis & treatment available




May cause severe human disease,

Serious Hazard to laboratory workers

Pose a risk to spread in community,

Effective prophylaxis & treatment available



Cause severe human disease,

Serious Hazard to laboratory workers

Pose a high risk to spread in community,

No Effective prophylaxis & treatment


Ebola, Marburg, Lassa, variola are pathogens of hazard group 4

Test Your Skills Now!
Take a Quiz now
Reviewer Name