Microbiology सूक्ष्मजीवशास्त्र

Quick summary

A healthy human body carries trillions of microorganisms on the skin and in the mouth, gut, upper respiratory tract and genital tract. This normal flora (microbiota) usually helps us, but some members can cause disease when they reach the wrong place or when immunity falls. Nosocomial (hospital-acquired) infections are infections that patients pick up in hospitals; they are a major problem because hospital organisms are often antibiotic-resistant.

Key points

• Resident flora live permanently at a site; transient flora stay for hours to days. Internal organs, blood, CSF and the lower respiratory tract are normally sterile.
• Skin: Staphylococcus epidermidis (most common), S. aureus (nose and skin of some people), Cutibacterium (Propionibacterium) acnes in hair follicles, diphtheroids, Malassezia yeast.
• Mouth: viridans streptococci (Streptococcus mutans causes dental caries), Actinomyces, anaerobes like Fusobacterium; plaque is a biofilm.
• Upper respiratory tract: viridans streptococci, Neisseria (non-pathogenic), Haemophilus; carriers may hold S. pneumoniae or S. aureus.
• Stomach: few organisms due to acid (pH about 2); Helicobacter pylori in some people.
• Large intestine: the densest population (about 10¹¹ bacteria per gram of contents), over 99% anaerobes — Bacteroides, Bifidobacterium, Clostridium, Eubacterium; plus E. coli, Enterococcus and others.
• Vagina: Lactobacillus (Döderlein's bacilli) after puberty keep pH acidic (about 4–4.5) by making lactic acid from glycogen.
• Benefits of normal flora: synthesis of vitamin K and B vitamins, colonization resistance (block pathogens by competing for space and food and making bacteriocins), training of the immune system, digestion of complex carbohydrates.
• Harm: opportunistic infections when flora move to other sites (E. coli → urinary tract infection) or when the host is weak (Candida in diabetes and HIV). Antibiotics can disturb the flora and allow Clostridioides (Clostridium) difficile to cause pseudomembranous colitis.
• Germ-free (gnotobiotic) animals show poorly developed immunity, proving the role of normal flora.
• Nosocomial infection = infection not present or incubating at admission, appearing 48 hours or more after admission (or within 30 days of surgery). Now often called healthcare-associated infection (HAI).
• Common types: urinary tract infection (often catheter-associated), surgical site infection, ventilator-associated pneumonia, bloodstream infection (central-line), and gastrointestinal infection.
• Common organisms: MRSA (methicillin-resistant S. aureus), Pseudomonas aeruginosa, Klebsiella pneumoniae and E. coli (ESBL and carbapenem-resistant strains), Acinetobacter baumannii, Enterococcus (VRE), Candida.
• Sources and spread: patient's own flora (endogenous), staff hands (most important route), contaminated instruments, catheters, air and water.
• Risk factors: long hospital stay, ICU, surgery, catheters and ventilators, old age, newborns, diabetes, cancer treatment, overuse of antibiotics.
• Prevention (infection control): hand hygiene (WHO's five moments; alcohol hand rub), sterilization of instruments, aseptic technique, isolation of infected patients, proper biomedical waste disposal, an antibiotic policy, surveillance by a hospital infection control committee.

Important terms

• Normal flora (microbiota) – microbes that normally live on or in a healthy body.
• Opportunistic pathogen – microbe that causes disease only when host defences are weak or it reaches an unusual site.
• Colonization resistance – protection given by normal flora against incoming pathogens.
• Nosocomial infection – infection acquired in hospital.
• MRSA – Staphylococcus aureus resistant to methicillin and most β-lactam antibiotics.

Key facts

5 important questions

Q1. What are opportunistic pathogens? Give an example.

Microbes that normally do not cause disease but do so when host immunity is low or when they reach another body site. Example: Candida albicans causing thrush in HIV patients, or E. coli causing urinary tract infection.

Q2. Define nosocomial infection.

An infection acquired in a hospital that was neither present nor incubating at admission, usually appearing 48 hours or more after admission.

Q3. Describe the normal flora of the human body and its significance.

• Sterile sites vs colonized sites.
• Flora of skin, mouth, respiratory tract, intestine, vagina (table).
• Beneficial roles and harmful roles.
• Effect of antibiotics; gnotobiotic animals.

Q4. Explain the sources, common organisms and types of hospital-acquired infections.

• Endogenous and exogenous sources; role of hands and devices.
• Types: CAUTI, SSI, VAP, CLABSI.
• Organisms: MRSA, Pseudomonas, Klebsiella, Acinetobacter, VRE.
• Risk factors.

Q5. How can nosocomial infections be prevented?

• Hand hygiene and personal protective equipment.
• Sterilization, disinfection and aseptic procedures.
• Isolation and surveillance.
• Antibiotic stewardship.
• Waste management and staff training.

Exam tip

Learn the five main HAI types with abbreviations and the five organisms most often named. "Hand washing is the single most important preventive measure" is a line worth writing.

Quick summary

Many important human diseases are caused by bacteria. This topic covers the general ideas of pathogenicity and virulence, then the causative agent, transmission, symptoms, laboratory diagnosis, treatment and prevention of key bacterial diseases taught in BSc: tuberculosis, typhoid, cholera, diphtheria, tetanus, leprosy and others.

Key points

• Pathogenicity is the ability to cause disease; virulence is the degree of pathogenicity.
• Virulence factors: capsules, adhesins (pili), invasive enzymes (hyaluronidase, coagulase), exotoxins (secreted proteins, very potent, e.g. tetanus and diphtheria toxins) and endotoxins (LPS of Gram-negative walls; cause fever and shock).
• Tuberculosis: Mycobacterium tuberculosis (Robert Koch, 1882). Acid-fast rod. Spread by droplets. Symptoms: cough for over 2 weeks, fever, night sweats, weight loss, blood in sputum. Diagnosis: sputum ZN staining, culture on Lowenstein–Jensen medium, chest X-ray, CBNAAT/GeneXpert, Mantoux (tuberculin) test. Treatment: combination drugs (isoniazid, rifampicin, pyrazinamide, ethambutol) under DOTS. Prevention: BCG vaccine.
• Typhoid (enteric fever): Salmonella enterica serovar Typhi. Spread by contaminated food and water (faeco-oral); carriers (e.g. "Typhoid Mary"). Step-ladder fever, abdominal pain, rose spots. Diagnosis: blood culture (first week), Widal test (agglutination, from second week), stool culture later. Treatment: antibiotics such as ceftriaxone or azithromycin (resistance is common). Prevention: safe water, sanitation, typhoid vaccine.
• Cholera: Vibrio cholerae (O1 and O139). Comma-shaped, motile. Faeco-oral. Cholera toxin causes profuse "rice-water" stools and severe dehydration. Treatment: ORS and IV fluids are life-saving; antibiotics (doxycycline) shorten illness. Diagnosis: stool culture on TCBS agar (yellow colonies).
• Diphtheria: Corynebacterium diphtheriae, Gram-positive club-shaped rods with metachromatic granules (Albert stain). Grey pseudomembrane in throat; exotoxin damages heart and nerves. Treatment: antitoxin + antibiotics. Prevention: DPT vaccine.
• Tetanus: Clostridium tetani, anaerobic spore-former ("drumstick" spores). Enters through deep wounds. Tetanospasmin causes muscle spasms, lockjaw, risus sardonicus. Prevention: tetanus toxoid; wound care; anti-tetanus immunoglobulin.
• Leprosy (Hansen's disease): Mycobacterium leprae (Hansen, 1873). Cannot be grown on artificial media (grows in armadillo and mouse footpad). Affects skin and peripheral nerves; tuberculoid and lepromatous types. Treatment: multidrug therapy (dapsone, rifampicin, clofazimine).
• Other bacterial diseases: plague (Yersinia pestis), pneumonia (Streptococcus pneumoniae), gonorrhoea (Neisseria gonorrhoeae), syphilis (Treponema pallidum), whooping cough (Bordetella pertussis), urinary tract infections (E. coli).

Important terms

• Exotoxin – protein toxin secreted by bacteria.
• Endotoxin – lipid A part of LPS in Gram-negative walls.
• Toxoid – toxin made harmless but still immunogenic (used in vaccines).
• Carrier – infected person with no symptoms who spreads the pathogen.
• Incubation period – time from infection to first symptoms.

Key facts

5 important questions

Q1. Differentiate exotoxin and endotoxin.

Exotoxins are heat-labile proteins secreted mainly by Gram-positive bacteria, highly potent and convertible to toxoids. Endotoxins are heat-stable lipopolysaccharides (lipid A) of Gram-negative walls, released when cells die, less potent and not convertible to toxoids.

Q2. What is the Widal test?

A tube or slide agglutination test that detects antibodies against the O and H antigens of Salmonella Typhi and Paratyphi in the patient's serum; used in diagnosis of typhoid from the second week.

Q3. Describe tuberculosis under: causative agent, transmission, symptoms, diagnosis, treatment, prevention.

• Agent: M. tuberculosis, acid-fast, slow-growing.
• Droplet spread.
• Symptoms as above; primary complex in lungs.
• Diagnosis: ZN stain, LJ culture, GeneXpert, X-ray, Mantoux.
• Treatment: multi-drug under DOTS, 6 months minimum.
• Prevention: BCG, early detection, ventilation, nutrition.

Q4. Write an account of cholera.

• V. cholerae: Gram-negative, comma-shaped, monotrichous.
• Faeco-oral via water.
• Toxin raises cAMP in gut cells → loss of water and salts.
• Rice-water stools, dehydration, shock.
• Diagnosis: hanging drop (darting motility), TCBS culture.
• Treatment: ORS, IV fluids, antibiotics.
• Prevention: safe water, sanitation, oral vaccine.

Q5. Describe tetanus.

• C. tetani: Gram-positive, anaerobic, terminal round spores.
• Spores in soil enter deep wounds.
• Tetanospasmin blocks inhibitory neurotransmitters → spastic paralysis, lockjaw.
• Neonatal tetanus from unclean cord cutting.
• Treatment: wound cleaning, anti-tetanus immunoglobulin, sedatives.
• Prevention: tetanus toxoid (in DPT, TT/Td boosters).

Exam tip

For each disease, answer under fixed headings — agent, transmission, symptoms, diagnosis, treatment, prevention. This format fetches full marks.

Quick summary

Viruses cause many important human diseases, from the common cold to AIDS. This topic covers how viruses cause disease and then the virus, spread, symptoms, diagnosis and prevention of key viral diseases in the BSc syllabus: AIDS, hepatitis, polio, rabies, influenza, measles, dengue and COVID-19.

Key points

• Viral infection steps: entry → attachment to specific receptors → multiplication inside cells → spread → cell damage and immune response → shedding to new hosts.
• Antibiotics do not work on viruses. Antiviral drugs exist for some (HIV, herpes, influenza, hepatitis B and C); vaccines are the main prevention.
• AIDS: caused by HIV (human immunodeficiency virus), an enveloped retrovirus with two copies of ssRNA and reverse transcriptase. It infects CD4⁺ T helper cells (via CD4 and co-receptors CCR5/CXCR4). Spread by unprotected sex, infected blood and needles, mother to child. Leads to fall in CD4 count and opportunistic infections (TB, Candida, Pneumocystis pneumonia). Diagnosis: ELISA/rapid tests for antibodies, confirmed by further tests; viral load by PCR. Treatment: antiretroviral therapy (ART); no vaccine yet.
• Hepatitis A and E: faeco-oral (contaminated water/food); usually self-limiting; vaccine for A.
• Hepatitis B: DNA virus; spread by blood, sex, mother to child; can become chronic → cirrhosis, liver cancer; HBsAg test for diagnosis; effective vaccine.
• Hepatitis C: RNA virus; blood-borne; often chronic; curable with modern antivirals; no vaccine.
• Hepatitis: inflammation of liver; jaundice.
• Polio (poliomyelitis): poliovirus, a small +ssRNA picornavirus (enterovirus). Faeco-oral; may attack motor neurons in the spinal cord → flaccid paralysis. Vaccines: Salk (IPV, injected inactivated) and Sabin (OPV, oral live attenuated). India was declared polio-free by WHO in 2014.
• Rabies: rabies virus (bullet-shaped, −ssRNA, rhabdovirus). Spread by bites of infected dogs (main source in India) and other mammals. Travels along nerves to the brain; symptoms include hydrophobia (fear of water); nearly always fatal once symptoms start. Negri bodies in brain cells. Prevention: wash wound with soap and water, post-exposure vaccination and rabies immunoglobulin; dog vaccination. Pasteur made the first rabies vaccine (1885).
• Influenza: orthomyxovirus, segmented −ssRNA, with haemagglutinin (H) and neuraminidase (N) spikes; antigenic drift (small changes) and shift (major changes → pandemics). Droplet spread. Antiviral: oseltamivir. Annual vaccine.
• Measles: paramyxovirus; droplets; fever, rash, Koplik's spots in mouth; MMR/MR vaccine.
• Dengue: flavivirus; spread by Aedes aegypti mosquito; high fever, severe body pain ("breakbone fever"), low platelets; severe dengue can cause bleeding and shock. Diagnosis: NS1 antigen, IgM ELISA. Prevention: mosquito control.
• COVID-19: SARS-CoV-2, enveloped +ssRNA coronavirus with spike protein binding ACE2 receptor; droplets and aerosols; diagnosed by RT-PCR and rapid antigen tests; vaccines used in India included Covishield and Covaxin.

Important terms

• Retrovirus – RNA virus that makes DNA from its RNA using reverse transcriptase.
• Opportunistic infection – infection by microbes that harm mainly people with weak immunity.
• Attenuated vaccine – vaccine with live but weakened virus.
• Antigenic shift / drift – major / minor changes in surface antigens.
• Vector – organism that carries a pathogen (e.g. mosquito).

Key facts

5 important questions

Q1. Why are antibiotics not effective against viruses?

Antibiotics act on bacterial structures like the cell wall, 70S ribosomes and bacterial enzymes. Viruses lack these and use the host cell's machinery, so antibiotics have no target in them.

Q2. Differentiate Salk and Sabin polio vaccines.

Salk vaccine (IPV) contains inactivated (killed) virus and is injected. Sabin vaccine (OPV) contains live attenuated virus and is given orally; it also gives gut immunity.

Q3. Describe AIDS under: agent, structure, transmission, pathogenesis, diagnosis and prevention.

• HIV: enveloped retrovirus; gp120 and gp41; two ssRNA strands; reverse transcriptase, integrase, protease.
• Transmission routes.
• Infects CD4⁺ T cells → their decline → immune failure.
• Stages: acute, latent, AIDS.
• Diagnosis: ELISA/rapid tests, confirmation, viral load.
• Prevention: safe sex, screened blood, sterile needles, ART for mothers; treatment by ART.

Q4. Write a note on rabies.

• Rhabdovirus, bullet-shaped, −ssRNA.
• Bite of rabid dog; virus in saliva.
• Moves along nerves to brain (long, variable incubation).
• Symptoms: fever, hydrophobia, agitation, paralysis, death.
• Diagnosis: Negri bodies, fluorescent antibody test.
• Post-exposure prophylaxis: wound washing, vaccine, immunoglobulin.

Q5. Compare hepatitis A and hepatitis B.

• Virus: RNA vs DNA.
• Spread: faeco-oral vs blood/sex/mother–child.
• Incubation: shorter vs longer.
• Chronic stage: no vs possible (cirrhosis, cancer).
• Marker: anti-HAV IgM vs HBsAg.
• Vaccine available for both.

Exam tip

Learn the genome type of each virus (DNA/RNA, + or −) — it is a common one-mark and short-answer point.

Quick summary

Fungi and protozoa cause many important human diseases in India — from common ringworm and thrush to life-threatening malaria, kala-azar and black fungus (mucormycosis, seen widely during COVID-19 in 2021). Mycoses are fungal diseases and are grouped by depth of infection. Protozoal diseases often spread through mosquitoes, sand flies or contaminated food and water. This topic covers the main agents, transmission, symptoms, laboratory diagnosis and control.

Key points

• Superficial – outer skin and hair, no inflammation: pityriasis versicolor (Malassezia furfur).
• Cutaneous – skin, hair, nails: dermatophytosis (ringworm, tinea) by Trichophyton, Microsporum, Epidermophyton; spread by contact, towels, combs.
• Subcutaneous – through wounds: mycetoma (Madura foot), sporotrichosis.
• Systemic (deep) – inhaled spores; histoplasmosis, aspergillosis, cryptococcosis.
• Opportunistic – in weak hosts: candidiasis (Candida albicans: oral thrush, vaginal infection), cryptococcal meningitis (Cryptococcus neoformans, capsulated yeast, seen with India ink) in AIDS, aspergillosis, mucormycosis (Rhizopus, Mucor; in uncontrolled diabetes and steroid use).
• Classification of mycoses:
• Lab diagnosis of fungi: KOH mount (10–20% KOH dissolves keratin, shows hyphae), culture on Sabouraud dextrose agar, germ tube test for C. albicans, India ink for Cryptococcus, serology.
• Antifungals: azoles (fluconazole), polyenes (amphotericin B – drug of choice for mucormycosis), griseofulvin and terbinafine (ringworm), nystatin (topical).
• Life cycle: sporozoites from mosquito → liver (exo-erythrocytic stage) → merozoites infect RBCs → ring, trophozoite, schizont → RBC bursts → fever cycles (48 h for vivax/falciparum, 72 h for malariae). Gametocytes taken up by mosquito → sexual cycle in mosquito.
• P. vivax and P. ovale form hypnozoites (dormant liver forms) → relapse; primaquine needed.
• Diagnosis: thick and thin peripheral blood smear (Giemsa/Leishman stain), rapid antigen tests.
• Treatment: chloroquine (vivax), artemisinin-based combination therapy (ACT) for falciparum.
• Malaria – Plasmodium vivax, P. falciparum (most dangerous; cerebral malaria), P. malariae, P. ovale (and P. knowlesi); vector **female Anopheles mosquito**.
• Amoebiasis – Entamoeba histolytica; faeco-oral spread through cysts (4 nuclei) in water/food; amoebic dysentery (blood and mucus), amoebic liver abscess; trophozoites contain ingested RBCs. Drug: metronidazole.
• Giardiasis – Giardia lamblia (intestinalis); flagellate with "falling leaf" motility; fatty diarrhoea and malabsorption; water-borne.
• Kala-azar (visceral leishmaniasis) – Leishmania donovani; vector sand fly (Phlebotomus); fever, enlarged spleen and liver, darkened skin; endemic in Bihar, Jharkhand, West Bengal; LD bodies (amastigotes) in bone marrow/spleen; rK39 rapid test.
• Toxoplasmosis – Toxoplasma gondii; from cat faeces and undercooked meat; dangerous in pregnancy (congenital infection) and in AIDS.
• Trichomoniasis – Trichomonas vaginalis; sexually transmitted flagellate.
• Control of protozoal diseases: vector control (bed nets, insecticide spraying, removing breeding sites), safe water and sanitation, early diagnosis and treatment; India's National Vector Borne Disease Control Programme (NVBDCP), now run by the National Center for Vector Borne Diseases Control.

Important terms

• Mycosis – disease caused by a fungus.
• Dermatophyte – fungus that infects keratin (skin, hair, nails).
• Hypnozoite – dormant liver stage of P. vivax and P. ovale causing relapse.
• Cyst – resistant, infective stage of intestinal protozoa.
• Vector – insect that transmits a parasite (mosquito, sand fly).

Key facts

5 important questions

Q1. What is the KOH mount used for?

It is a quick test for fungal infection of skin, hair and nails. KOH (10–20%) dissolves keratin and cells, so fungal hyphae or spores can be seen clearly under the microscope.

Q2. Why do P. vivax infections relapse?

Because P. vivax forms hypnozoites, dormant stages in the liver that can wake up weeks or months later and release merozoites into blood. Primaquine kills them.

Q3. Classify mycoses with examples and describe candidiasis.

• Superficial, cutaneous, subcutaneous, systemic, opportunistic with examples.
• Candida albicans: yeast and pseudohyphae; thrush, vaginitis, systemic candidiasis.
• Predisposing factors, diagnosis, treatment.

Q4. Describe the life cycle of malarial parasite and lab diagnosis of malaria.

• Human cycle (liver and RBC stages, schizogony).
• Mosquito cycle (gametogony and sporogony).
• Fever periodicity.
• Diagnosis by thick and thin smear and rapid tests; treatment.

Q5. Write notes on amoebiasis and kala-azar.

• E. histolytica: cysts, trophozoites, dysentery, liver abscess, diagnosis, metronidazole.
• L. donovani: sand fly, symptoms, LD bodies, rK39, treatment (miltefosine, liposomal amphotericin B), elimination programme.

Exam tip

Draw the malaria life cycle clearly — it is the most repeated long question. For fungi, remember "KOH for skin, India ink for Cryptococcus, germ tube for Candida".

Quick summary

Genetic engineering (recombinant DNA technology) is the deliberate change of an organism's DNA by cutting, joining and transferring genes. This topic covers the tools — restriction enzymes, ligase, vectors and hosts — the steps of gene cloning, screening methods, PCR, and applications such as insulin production and Bt crops.

Key points

• The first recombinant DNA was made by Stanley Cohen and Herbert Boyer (1973).
• Restriction endonucleases ("molecular scissors") cut DNA at specific palindromic recognition sequences. Example: EcoRI (from E. coli) cuts G↓AATTC, leaving sticky ends (AATT overhangs). Some enzymes (e.g. SmaI) give blunt ends.
• Naming: EcoRI = E (genus Escherichia) + co (species coli) + R (strain RY13) + I (first enzyme found).
• DNA ligase ("molecular glue") joins DNA fragments by forming phosphodiester bonds.
• Vectors carry the gene into the host. Good vector features: origin of replication (ori), selectable marker (e.g. antibiotic resistance), unique restriction sites (multiple cloning site), small size.
• Types of vectors: plasmids (pBR322 — ampicillin and tetracycline resistance genes; pUC vectors with lacZ for blue-white screening), bacteriophage lambda, cosmids, BACs, YACs, Ti plasmid of Agrobacterium tumefaciens (for plants), viral vectors (for animal cells).
• Hosts: E. coli (most common), Bacillus subtilis, yeast, plant and animal cells.
• Steps of gene cloning: isolate gene of interest → cut gene and vector with the same restriction enzyme → join with ligase → introduce into host (transformation by CaCl₂/heat shock or electroporation) → select and screen recombinants → multiply and express.
• Screening: insertional inactivation — e.g. gene inserted in the tetracycline gene of pBR322 makes recombinants ampicillin-resistant but tetracycline-sensitive (checked by replica plating); blue-white screening — insert disrupts lacZ, so recombinant colonies are white on X-gal, non-recombinants blue.
• PCR (Kary Mullis, 1983) amplifies a DNA segment millions of times. Each cycle: denaturation (about 94–95 °C), annealing of primers (about 50–65 °C), extension (72 °C) by heat-stable Taq polymerase (from Thermus aquaticus).
• Other tools: gel electrophoresis (separates DNA by size), Southern blotting (DNA), Northern (RNA), Western (protein), DNA sequencing (Sanger method), CRISPR-Cas9 gene editing.
• Applications: human insulin made in E. coli (first recombinant drug approved, 1982, Humulin), human growth hormone, hepatitis B vaccine made in yeast, Bt cotton (cry genes from Bacillus thuringiensis; approved in India in 2002), Golden Rice, gene therapy, DNA fingerprinting, diagnostic kits.
• Biosafety and ethics: regulated in India by bodies such as GEAC (Genetic Engineering Appraisal Committee).

Important terms

• Recombinant DNA – DNA formed by joining DNA from different sources.
• Palindrome – DNA sequence that reads the same 5′→3′ on both strands.
• Sticky ends – short single-stranded overhangs after cutting.
• Selectable marker – gene that allows selection of transformed cells.
• cDNA – DNA copied from mRNA by reverse transcriptase (no introns).

Formulas

• PCR amplification: number of copies = 2ⁿ (from one double-stranded target, ideal), n = number of cycles.
• Example: after 30 cycles, 2³⁰ ≈ 1.07 × 10⁹ copies (about one billion).
• After 10 cycles: 2¹⁰ = 1024 copies.

5 important questions

Q1. What are restriction enzymes? Give an example.

Enzymes that recognise specific short DNA sequences and cut both strands at or near them. Example: EcoRI cuts between G and A in GAATTC, producing sticky ends.

Q2. How many copies of DNA are made from one molecule after 20 PCR cycles?

Copies = 2²⁰ = 1,048,576 ≈ 10⁶ copies (ideal conditions).

Q3. Explain the steps of gene cloning.

• Isolation of DNA and gene of interest.
• Cutting with restriction enzyme; vector cut with same enzyme.
• Ligation → recombinant DNA.
• Transformation of host (E. coli).
• Selection on antibiotic medium.
• Screening (blue-white/insertional inactivation).
• Culture and expression of the product.

Q4. Describe the features and use of pBR322.

• Small E. coli plasmid, about 4.4 kb (4361 bp).
• ori for replication.
• ampR and tetR genes as markers.
• Unique restriction sites (e.g. BamHI and SalI in tetR; PstI in ampR; EcoRI site).
• Insertional inactivation screening explained.
• Named after Bolivar and Rodriguez (p = plasmid, BR = their initials).

Q5. Explain the polymerase chain reaction.

• Components: template DNA, two primers, dNTPs, Taq polymerase, buffer with Mg²⁺.
• Denaturation at 94–95 °C.
• Annealing at 50–65 °C.
• Extension at 72 °C.
• 25–35 cycles in a thermal cycler; exponential amplification (2ⁿ).
• Uses: diagnosis (COVID-19 RT-PCR), forensics, cloning, research.

Exam tip

Draw the cloning flowchart and the pBR322 map with ori, ampR and tetR marked — both are frequently asked.

Quick summary

Modern microbiology depends on a few powerful molecular techniques for detecting and studying DNA, RNA and proteins. PCR makes millions of copies of a chosen DNA piece in a few hours, blotting methods find a specific DNA, RNA or protein among thousands, and DNA sequencing reads the exact order of bases. These tools are used in diagnosis (COVID-19 RT-PCR, TB, HIV), forensic DNA fingerprinting, genetic engineering and microbial identification (16S rRNA sequencing).

Key points

• PCR (polymerase chain reaction) was invented by Kary Mullis (1983); Nobel Prize in Chemistry 1993.
• Ingredients: template DNA, two primers (short single-stranded DNA, about 18–25 bases, flanking the target), dNTPs (dATP, dGTP, dCTP, dTTP), heat-stable Taq DNA polymerase (from Thermus aquaticus, a hot-spring bacterium), buffer with Mg²⁺.
• Denaturation at about 94–95 °C – DNA strands separate.
• Annealing at about 50–65 °C – primers bind to complementary sites.
• Extension at 72 °C – Taq polymerase builds new strands 5′ → 3′.
• One PCR cycle (in a thermal cycler):
• Each cycle doubles the target; after n cycles about 2ⁿ copies (30 cycles ≈ 10⁹ copies). Products are checked by agarose gel electrophoresis.
• Variants: RT-PCR (reverse transcriptase first copies RNA to cDNA — used for RNA viruses like SARS-CoV-2 and HIV load), real-time / quantitative PCR (qPCR) (fluorescent dye or probe measures product as it forms; Ct value), multiplex PCR (several targets at once), nested PCR.
• Applications of PCR: diagnosis of infections (TB – GeneXpert, COVID-19, HIV, hepatitis), DNA fingerprinting, paternity tests, cloning genes, mutation detection, detecting GMOs and food pathogens.
• Southern blotting (Edwin Southern, 1975): DNA cut by restriction enzymes → separated by agarose gel electrophoresis → denatured and transferred to a nitrocellulose/nylon membrane → hybridized with a labelled DNA probe → detected by autoradiography or colour. Used in RFLP analysis and DNA fingerprinting.
• Northern blotting: same idea for RNA (detects gene expression).
• Western blotting (immunoblot): proteins separated by SDS-PAGE → transferred to membrane → detected with specific antibodies. Used as a confirmatory test for HIV antibodies in the past.
• Sanger's dideoxy (chain termination) method (Frederick Sanger, 1977): uses ddNTPs (no 3′-OH), which stop chain growth; fragments of different lengths are separated and read. Modern versions use fluorescent ddNTPs and capillary electrophoresis.
• Maxam–Gilbert method (chemical cleavage, 1977) is now mostly historical.
• Next-generation sequencing (NGS) reads millions of fragments in parallel (Illumina etc.); used for whole-genome sequencing of pathogens and outbreak tracking.
• DNA sequencing:
• 16S rRNA gene sequencing identifies bacteria and studies microbial communities (metagenomics).
• DNA fingerprinting in India was developed by Lalji Singh at CCMB, Hyderabad (CDFD later set up for it).

Important terms

• Primer – short single-stranded DNA that starts DNA synthesis at a specific site.
• Taq polymerase – heat-stable DNA polymerase from Thermus aquaticus.
• Probe – labelled DNA or RNA piece that finds and binds a complementary sequence.
• Hybridization – pairing of complementary nucleic acid strands.
• Dideoxynucleotide (ddNTP) – nucleotide lacking 3′-OH that stops chain extension.

Formulas

• Copies after n cycles ≈ N₀ × 2ⁿ (N₀ = starting copies).
• Example: 10 starting copies, 20 cycles → 10 × 2²⁰ = 10 × 1,048,576 ≈ 1.05 × 10⁷ copies.

5 important questions

Q1. Why is Taq polymerase used in PCR?

Because it comes from the thermophile Thermus aquaticus and is not destroyed at the 94–95 °C denaturation step, so it does not have to be added again in every cycle; its best working temperature is about 72 °C.

Q2. What is RT-PCR?

Reverse transcription PCR: reverse transcriptase first makes complementary DNA (cDNA) from an RNA template, which is then amplified by PCR. It is used to detect RNA viruses such as SARS-CoV-2 and to study gene expression.

Q3. Explain the principle, steps and applications of PCR.

• Components.
• Three steps with temperatures; exponential amplification (diagram).
• Variants (RT-PCR, qPCR, multiplex).
• Applications in diagnosis, forensics, research.

Q4. Describe Southern blotting and compare it with Northern and Western blotting.

• Steps from digestion to detection (flow chart).
• Uses: RFLP, DNA fingerprinting, gene detection.
• Comparison table of three blots.

Q5. Explain Sanger's dideoxy method of DNA sequencing.

• Template, primer, DNA polymerase, dNTPs and a small amount of ddNTPs.
• Chain termination at each base.
• Four reactions or four fluorescent dyes; gel/capillary reading.
• Brief mention of NGS and its use in microbiology.

Exam tip

Memorise the three PCR temperatures (95, 55, 72 °C) and the "S-N-W = DNA-RNA-Protein" rule for blots (Southern, Northern, Western).

Quick summary

Environmental microbiology studies microbes in air, water, soil and extreme habitats and their role in the environment. This topic covers biogeochemical cycles (carbon, sulphur, phosphorus), air microflora, biodegradation and bioremediation, biogas production, biological leaching, and microbes as tools for pollution control.

Key points

• Microbes are the main decomposers: they break dead matter into simple substances and recycle elements.
• Carbon cycle: CO₂ fixed by plants, algae and cyanobacteria (photosynthesis) and chemoautotrophs; released by respiration and decomposition. Methanogens (archaea) produce methane in anaerobic places (paddy fields, cattle rumen, swamps); methanotrophs oxidize methane.
• Sulphur cycle: decomposers release H₂S from proteins; sulphur-oxidizing bacteria (Thiobacillus, Beggiatoa) oxidize H₂S/S to sulphate; sulphate-reducing bacteria (Desulfovibrio) reduce sulphate to H₂S under anaerobic conditions.
• Phosphorus cycle: no gas phase; microbes mineralize organic phosphate and solubilize rock phosphate.
• Air microbiology: air has no native flora; microbes arrive as droplets, dust and spores. Common: spores of Aspergillus, Penicillium, Cladosporium, bacteria like Bacillus, Micrococcus. Air-borne diseases: TB, influenza, measles, COVID-19.
• Air sampling: settle plate (gravity) method, slit sampler, impinger, filtration.
• Biodegradation: breakdown of substances by microbes. Natural materials are biodegradable; many plastics and pesticides (DDT) are recalcitrant (resist breakdown).
• Bioremediation: using microbes to clean polluted sites. In situ (at site; e.g. bioventing, biostimulation, bioaugmentation) and ex situ (removed for treatment; e.g. composting, land farming, bioreactors).
• Example: Pseudomonas putida "superbug" engineered by Ananda Mohan Chakrabarty (1970s) to degrade several hydrocarbons in oil; it was the first patented living organism (US Supreme Court case, 1980).
• Phytoremediation uses plants (with root microbes) to remove pollutants.
• Biogas: anaerobic digestion of cow dung and organic waste in three stages — hydrolysis, acidogenesis/acetogenesis (acids, H₂, CO₂), methanogenesis (methanogens like Methanobacterium make CH₄). Biogas is about 50–70% methane plus CO₂. Slurry is good manure. Common Indian plants: KVIC floating-drum type and Deenbandhu fixed-dome type.
• Bioleaching: Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans) oxidizes metal sulphide ores, releasing copper, uranium etc. from low-grade ores.
• Acid mine drainage occurs when such bacteria oxidize sulphide minerals in mine waste, forming sulphuric acid.
• Extremophiles: thermophiles (Thermus aquaticus in hot springs), halophiles (Halobacterium in salt pans), acidophiles, psychrophiles.
• Biopesticides: Bacillus thuringiensis (Bt) toxin kills insect larvae; Trichoderma fights fungal plant pathogens.

Important terms

• Biogeochemical cycle – movement of an element between living things and the environment.
• Bioremediation – clean-up of pollutants by living organisms.
• Recalcitrant / xenobiotic – hard to degrade / man-made foreign chemical.
• Bioaugmentation – adding specific microbes to a polluted site.
• Biostimulation – adding nutrients or oxygen to boost native microbes.

Formulas

• Methanogenesis from CO₂: CO₂ + 4H₂ → CH₄ + 2H₂O
• Methanogenesis from acetate: CH₃COOH → CH₄ + CO₂
• Sulphide oxidation (overall): H₂S + 2O₂ → SO₄²⁻ + 2H⁺
• Sulphate reduction (overall): SO₄²⁻ + 4H₂ + 2H⁺ → H₂S + 4H₂O

5 important questions

Q1. What is bioremediation?

The use of microorganisms (or plants) to break down or remove pollutants like oil, pesticides and heavy metals from soil and water, turning them into less harmful substances.

Q2. Name two microbes involved in the sulphur cycle and their role.

Thiobacillus oxidizes sulphur and H₂S to sulphate; Desulfovibrio reduces sulphate to H₂S under anaerobic conditions.

Q3. Describe biogas production.

• Raw material: cow dung, kitchen and farm waste mixed with water.
• Anaerobic digester (fixed dome or floating drum).
• Hydrolysis: complex polymers → sugars, amino acids.
• Acidogenesis/acetogenesis: organic acids, H₂, CO₂.
• Methanogenesis: methanogens form CH₄.
• Gas 50–70% methane; used for cooking, lighting; slurry as manure.

Q4. Explain the types of bioremediation with examples.

• In situ: biostimulation, bioaugmentation, bioventing.
• Ex situ: land farming, composting, biopiles, bioreactors.
• Organisms: Pseudomonas (oil), Deinococcus radiodurans (radiation-tolerant, studied for radioactive waste), fungi like Phanerochaete chrysosporium (white-rot fungus).
• Advantages: cheap, eco-friendly; limitations: slow, site-specific.

Q5. Write a note on bioleaching.

• Microbial extraction of metals from ores.
• Organism: Acidithiobacillus ferrooxidans, A. thiooxidans.
• Bacteria oxidize Fe²⁺ to Fe³⁺ and sulphide to sulphuric acid.
• Fe³⁺ and acid dissolve metal sulphides (e.g. copper sulphide → soluble copper sulphate).
• Methods: dump/heap leaching, in situ leaching.
• Uses: copper, uranium, gold from low-grade ores.

Exam tip

Draw cycles with organism names on arrows, and write biogas stages in order with the microbe group responsible for each.

Quick summary

Extremophiles are microorganisms that grow best in conditions that would kill most living things — very high or low temperature, strong acid or alkali, high salt or high pressure. Many of them are archaea, but some are bacteria, fungi and algae. Their special proteins and membranes are adapted to these conditions, and their enzymes ("extremozymes") are valuable in industry and biotechnology, such as Taq polymerase used in PCR.

Key points

• Thermophiles: optimum about 45–80 °C (e.g. Geobacillus stearothermophilus, Thermus aquaticus – optimum about 70 °C, from Yellowstone hot springs, discovered by Thomas Brock).
• Hyperthermophiles: optimum above 80 °C (Pyrococcus furiosus about 100 °C; Pyrolobus fumarii grows up to about 113 °C; strain 121 reported growing at 121 °C). Found in hot springs and deep-sea hydrothermal vents. India has hot springs at Manikaran (Himachal Pradesh) and Unkeshwar (Maharashtra).
• Psychrophiles: optimum about 15 °C or lower, can grow at 0 °C (Polaromonas, Psychrobacter); psychrotrophs grow at 0–7 °C but have higher optima and spoil refrigerated food.
• Acidophiles: optimum pH below about 3 (Acidithiobacillus ferrooxidans in acid mine drainage; Picrophilus grows near pH 0).
• Alkaliphiles: optimum pH above about 9 (Bacillus spp. in soda lakes; Lonar crater lake in Buldhana district, Maharashtra, is an alkaline-saline lake with such microbes).
• Halophiles: need high salt; extreme halophiles need about 15–30% NaCl (Halobacterium salinarum, Haloferax; red colour in salt pans from carotenoids and bacteriorhodopsin).
• Barophiles (piezophiles): grow best under high pressure of the deep sea (Shewanella, Colwellia).
• Others: radioresistant Deinococcus radiodurans (survives very high radiation by efficient DNA repair), xerophiles (dry conditions), and polyextremophiles.
• Thermophiles: proteins with more ionic bonds and compact cores, heat-stable enzymes, reverse gyrase (positively supercoils DNA), high G + C rRNA, and archaeal ether-linked lipids (tetraether monolayer membranes).
• Psychrophiles: flexible enzymes active in cold, more unsaturated fatty acids keep membranes fluid, antifreeze and cold-shock proteins.
• Halophiles: "salt-in" strategy (accumulate K⁺ inside; acidic proteins) or "compatible solutes" (glycine betaine, ectoine) to balance osmotic pressure.
• Acidophiles: keep internal pH near neutral by pumping out H⁺; alkaliphiles use Na⁺ gradients.
• Adaptations:
• Taq polymerase and Pfu polymerase (from Pyrococcus furiosus, with proofreading) for PCR.
• Heat-stable amylases and proteases for starch processing and detergents (alkaline proteases from alkaliphiles).
• Cold-active enzymes for cold-water washing powders and food processing.
• Bioleaching of copper and uranium by Acidithiobacillus.
• Compatible solutes (ectoine) in cosmetics; bacteriorhodopsin in bioelectronics research.
• Bioremediation of extreme sites; studies of the origin of life and astrobiology.
• Applications:

Important terms

• Extremophile – organism that grows best in extreme conditions.
• Extremozyme – enzyme from an extremophile that works under extreme conditions.
• Compatible solute – small organic molecule accumulated by cells to balance high outside salt without harming enzymes.
• Bacteriorhodopsin – light-driven proton pump in the purple membrane of Halobacterium.
• Bioleaching – extraction of metals from ores using microbes.

Key facts

5 important questions

Q1. What are extremozymes? Give one example.

Enzymes from extremophiles that remain active under extreme temperature, pH or salt. Example: Taq DNA polymerase from Thermus aquaticus, which works at about 72 °C and survives 95 °C.

Q2. How do psychrophiles keep their membranes functional at low temperature?

They contain more unsaturated and short-chain fatty acids, which keep the membrane fluid in the cold. Their enzymes are also more flexible.

Q3. Classify extremophiles with examples.

• Based on temperature, pH, salt, pressure, radiation.
• Table with range and example organism.
• Indian habitats (hot springs, Lonar lake, salt pans of Gujarat).

Q4. Explain the molecular adaptations of thermophiles and halophiles.

• Thermophiles: protein stability, reverse gyrase, ether lipids, high G + C rRNA.
• Halophiles: salt-in strategy, compatible solutes, acidic proteins, S-layer walls, bacteriorhodopsin.

Q5. Describe the industrial applications of extremophiles.

• PCR enzymes (Taq, Pfu).
• Detergent and starch-processing enzymes.
• Bioleaching and bioremediation.
• Cosmetics, food and pharmaceuticals.

Exam tip

Give a temperature or pH range and an example for every extremophile type. Mentioning Lonar lake (Maharashtra) as an alkaline habitat makes your answer stand out.

Quick summary

Pharmaceutical microbiology deals with microbes in the making and testing of medicines. Microbes are used to produce drugs (antibiotics, vaccines, insulin, enzymes), and microbes must be kept out of or limited in medicines. Quality control (QC) tests check that injections are sterile, that oral products carry only safe numbers of microbes, that preservatives work, and that antibiotics have the right potency. The rules are written in the pharmacopoeia (for India, the Indian Pharmacopoeia) and in Good Manufacturing Practice (GMP).

Key points

• Sources of contamination: raw materials (natural products, water), air, equipment, packaging, and people (the biggest source — skin, hair, breath).
• Effects of contamination: spoilage (change in colour, smell, gas, loss of potency) and infection in patients, especially from contaminated eye drops and injections.
• Sterile products (injections, eye drops, IV fluids) must be free from all living microbes; non-sterile products (tablets, syrups, creams) have limits — e.g. absence of specified pathogens such as E. coli, Salmonella, Pseudomonas aeruginosa and Staphylococcus aureus depending on the product.
• Clean rooms: air filtered through HEPA filters; graded by number of particles (ISO classes; Grade A–D in GMP). Aseptic filling is done in Grade A (laminar flow) zones.
• Sterility test (Indian Pharmacopoeia / USP): by membrane filtration (0.45 µm filter, preferred) or direct inoculation; media fluid thioglycollate medium (anaerobes and aerobes, incubated at 30–35 °C) and soybean–casein digest medium (fungi and aerobes, 20–25 °C), incubated for 14 days. No growth = passes.
• Pyrogen / endotoxin test: pyrogens (mainly bacterial endotoxins, LPS) cause fever even after the bacteria are killed. Tested by the rabbit pyrogen test (older) or the LAL test (Limulus amoebocyte lysate from horseshoe crab blood clots in the presence of endotoxin; very sensitive). Pyrogens are removed from glassware by dry heat at about 250 °C for 30 minutes or more (depyrogenation).
• Microbial limit test for non-sterile products: total aerobic microbial count (TAMC) and total yeast and mould count (TYMC), plus tests for absence of specified organisms.
• Preservative efficacy (antimicrobial effectiveness) test: the product is challenged with known organisms (S. aureus, P. aeruginosa, E. coli, Candida albicans, Aspergillus brasiliensis) and counts are followed over 28 days.
• Cylinder-plate (cup-plate) method: diameter of zones of inhibition around cups/wells compared with a standard.
• Turbidimetric (tube) method: growth inhibition measured as turbidity.
• Vitamins (B₁₂) and amino acids are assayed with auxotrophic organisms (e.g. Lactobacillus leichmannii for B₁₂).
• Microbiological assay of antibiotics: potency measured by the antibiotic's effect on a sensitive test organism.
• Validation of sterilization: biological indicators — Geobacillus stearothermophilus spores for autoclave, Bacillus atrophaeus spores for dry heat and ethylene oxide, Bacillus pumilus spores for radiation; chemical indicators (autoclave tape) and physical records.
• Environmental monitoring: settle plates, air samplers, surface swabs and contact plates, and gloves/fingertip tests on operators.
• Regulators in India: CDSCO (Central Drugs Standard Control Organisation) under the Drugs and Cosmetics Act, 1940; GMP rules in Schedule M.

Important terms

• Pharmacopoeia – official book of standards for drugs and their tests.
• GMP – Good Manufacturing Practice: rules ensuring products are consistently made to quality standards.
• Pyrogen – fever-producing substance, mainly bacterial endotoxin.
• LAL test – endotoxin test using horseshoe crab blood cell extract.
• Biological indicator – standard spore preparation used to check sterilization.

Key facts

5 important questions

Q1. What is the LAL test?

A test for bacterial endotoxins (pyrogens) in injections. An extract of blood cells of the horseshoe crab (Limulus polyphemus) forms a gel or colour change when endotoxin is present. It is faster and more sensitive than the rabbit test.

Q2. Name the media used in the sterility test.

Fluid thioglycollate medium (for anaerobic and aerobic bacteria) and soybean–casein digest medium (for fungi and aerobic bacteria).

Q3. Describe the sterility testing of pharmaceutical products.

• Need and sampling.
• Membrane filtration method and direct inoculation method.
• Media, temperature, incubation time.
• Interpretation and controls (positive and negative).
• Done in aseptic clean-room conditions.

Q4. Explain microbiological assay of antibiotics.

• Principle: inhibition of sensitive organism proportional to concentration.
• Cylinder-plate method: seeded agar, cups, standard and test, zone measurement, standard curve.
• Turbidimetric method.
• Advantages over chemical assays (measures biological activity).

Q5. Write a note on sources of contamination and quality control in pharmaceutical industry.

• Sources: raw materials, water, air, personnel, equipment, packaging.
• GMP, clean rooms, environmental monitoring.
• QC tests (table).
• Role of Indian Pharmacopoeia and CDSCO.

Exam tip

Learn the two sterility test media with their incubation temperatures and the 14-day period. "LAL test" and "biological indicators" are favourite short notes.