Microbiology सूक्ष्मजीवशास्त्र

Quick summary

Food microbiology studies the microbes that spoil food, cause food-borne illness, and help make fermented foods. This topic covers factors affecting microbial growth in food, types of spoilage, food poisoning and food infection, methods of food preservation, and useful fermented foods including Indian examples.

Key points

• Intrinsic factors (inside food): pH, moisture (water activity, aw), nutrients, oxidation–reduction potential, natural antimicrobials (e.g. lysozyme in egg white), biological structure (shells, skins).
• Extrinsic factors (environment): storage temperature, relative humidity, gases (O₂, CO₂), presence of other microbes.
• Most bacteria need aw above about 0.90; moulds can grow at lower aw (around 0.80 or less); dried foods resist spoilage.
• Bread: moulds like Rhizopus stolonifer (black bread mould), Penicillium, Aspergillus; "rope" spoilage by Bacillus subtilis.
• Fruits and vegetables: soft rot by Erwinia (now Pectobacterium); moulds.
• Meat: putrefaction by Pseudomonas, Clostridium.
• Canned food: swelling due to gas from Clostridium; flat sour spoilage (acid without gas) by Geobacillus stearothermophilus.
• Spoilage signs: off-odour, sliminess, souring, gas, colour change, mould growth.
• Food poisoning (intoxication): illness from a toxin already formed in food — Staphylococcus aureus (heat-stable enterotoxin; vomiting within a few hours), Clostridium botulinum (botulinum neurotoxin; paralysis; linked to poorly canned low-acid foods), Bacillus cereus (reheated rice).
• Food infection: live pathogens eaten multiply in the gut — Salmonella (eggs, poultry), Shigella, E. coli O157:H7, Vibrio parahaemolyticus (seafood), Listeria monocytogenes (grows even at refrigerator temperature).
• Preservation methods: high temperature (pasteurization, canning, boiling), low temperature (refrigeration about 4 °C, freezing −18 °C or lower), drying/dehydration, high salt or sugar (lowers aw: pickles, jams), chemical preservatives (sodium benzoate, potassium metabisulphite, sorbic acid), irradiation, modified-atmosphere packaging, fermentation, bacteriocins like nisin (from Lactococcus lactis).
• Canning uses the 12-D concept: heat enough to reduce C. botulinum spores by 12 log cycles.
• Fermented foods: curd/dahi, idli and dosa batter (lactic acid bacteria such as Leuconostoc mesenteroides and yeasts), bread (Saccharomyces cerevisiae), vinegar (Acetobacter), sauerkraut, soy sauce, cheese.
• Probiotics: live beneficial microbes (e.g. Lactobacillus, Bifidobacterium) that improve health when eaten in enough amounts.

Important terms

• Water activity (aw) – free water available for microbes (pure water = 1.0).
• Intoxication – illness due to a pre-formed toxin.
• Food infection – illness due to growth of live pathogens in the body.
• Bacteriocin – antibacterial protein made by bacteria.
• Hurdle technology – combining several mild preservation methods.

Key facts

5 important questions

Q1. What is water activity?

The ratio of the vapour pressure of water in a food to that of pure water at the same temperature. It shows how much free water is available for microbial growth; lowering it (drying, salting) preserves food.

Q2. Differentiate food intoxication and food infection.

Intoxication is caused by eating toxin already formed in food (e.g. S. aureus, C. botulinum); symptoms start quickly. Infection is caused by live microbes multiplying in the body (e.g. Salmonella); symptoms take longer.

Q3. Explain intrinsic and extrinsic factors affecting microbial growth in food.

• Intrinsic: pH, aw, nutrients, Eh, antimicrobial constituents, biological structures.
• Extrinsic: temperature, humidity, gas atmosphere, competing microbes.
• Example: low pH of pickles and low aw of jams resist bacteria.
• These factors decide shelf life.

Q4. Describe methods of food preservation.

• Heat: pasteurization, sterilization, canning (12-D).
• Cold: refrigeration, freezing.
• Drying, smoking.
• Salt/sugar (osmotic effect).
• Chemicals: benzoates, sorbates, sulphites.
• Irradiation, nisin.
• Fermentation, packaging.

Q5. Write a note on botulism.

• Caused by Clostridium botulinum, Gram-positive, anaerobic, spore-forming rod.
• Toxin formed in low-acid, anaerobic foods (badly canned vegetables, meat).
• Neurotoxin blocks acetylcholine release → flaccid paralysis, double vision, breathing failure.
• Treatment: antitoxin, supportive care.
• Prevention: proper canning (12-D), boiling suspected food (toxin is heat-labile), discard swollen cans.

Exam tip

Learn organism – disease – food as a table; questions on food poisoning almost always ask for examples.

Quick summary

Fermented foods are foods changed by the controlled action of microorganisms, which improves taste, texture, shelf life and nutrition. India has a rich tradition of them — curd, idli, dosa, dhokla, jalebi batter, pickles. Probiotics are live beneficial microbes, prebiotics are the food that feeds them, and synbiotics combine both. This topic covers important fermented foods and the health role of probiotics.

Key points

• Lactic acid bacteria (LAB) — Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus thermophilus — are the main fermenting organisms. They are Gram-positive, non-spore-forming, catalase-negative and make lactic acid, which lowers pH and stops spoilage organisms.
• Yeasts (Saccharomyces cerevisiae) make CO₂ and ethanol (bread, beverages); moulds (Aspergillus oryzae, Rhizopus) are used in Asian foods like soy sauce and tempeh.
• Idli and dosa: soaked rice and black gram (urad dal) are ground and fermented overnight. Leuconostoc mesenteroides starts the fermentation and produces CO₂ that raises the batter; Enterococcus faecalis, Pediococcus and yeasts join later. The fermentation increases B vitamins and digestibility.
• Dhokla (Bengal gram batter), jalebi batter, kanji (black carrot drink), bhatura dough and dosa are other Indian fermented foods.
• Sauerkraut (fermented cabbage): heterofermentative Leuconostoc mesenteroides first, then homofermentative Lactobacillus plantarum; 2–3% salt draws out juice.
• Pickles (cucumber, mango) and olives are also LAB fermentations under salt.
• Bread: baker's yeast ferments sugars to CO₂ (raises dough) and ethanol (evaporates on baking).
• Vinegar: two steps — yeast changes sugar to ethanol, then Acetobacter (Gluconobacter) oxidizes ethanol to acetic acid (about 4–8%).
• Soy sauce (Aspergillus oryzae koji, then LAB and yeasts), tempeh (Rhizopus oligosporus), kimchi (Korean, LAB).
• Probiotics: "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" (FAO/WHO, 2001). Examples: Lactobacillus acidophilus, L. rhamnosus GG, Bifidobacterium spp., Saccharomyces boulardii (a yeast).
• Qualities of a good probiotic: survives stomach acid and bile, sticks to the gut lining, is safe (GRAS), has proven benefit, stays alive during storage.
• Benefits: prevent and treat diarrhoea (including antibiotic-associated diarrhoea), help lactose digestion, compete with pathogens, strengthen gut barrier and immunity, may lower cholesterol.
• Prebiotics: non-digestible food ingredients that feed good gut bacteria — inulin, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS); found in onion, garlic, banana, chicory.
• Synbiotics: probiotic + prebiotic together. Postbiotics: non-living microbial products or cell parts that give health benefits.

Important terms

• Starter culture – known microorganisms added to start a controlled fermentation.
• Homofermentative – LAB that make mainly lactic acid from glucose.
• Heterofermentative – LAB that make lactic acid plus CO₂ and ethanol or acetic acid.
• Probiotic – live microbe that gives health benefit in adequate amounts.
• Prebiotic – non-digestible food that selectively feeds beneficial gut microbes.

Key facts

5 important questions

Q1. What are prebiotics? Give two examples.

Prebiotics are non-digestible food ingredients that pass to the colon and selectively feed beneficial bacteria like bifidobacteria. Examples: inulin and fructo-oligosaccharides.

Q2. Why are lactic acid bacteria important in food fermentation?

They produce lactic acid that lowers pH, preserves the food, gives sour flavour, and some make bacteriocins (e.g. nisin) and vitamins. They are safe (GRAS).

Q3. Describe the microbiology of idli fermentation.

• Ingredients and soaking, grinding, overnight fermentation at room temperature.
• Role of Leuconostoc mesenteroides (CO₂, acid), later Enterococcus, Pediococcus, yeasts.
• Changes: leavening, pH fall, increased B vitamins and protein digestibility.
• Flow chart.

Q4. Explain probiotics: definition, properties and health benefits.

• FAO/WHO definition and examples.
• Selection criteria.
• Mechanisms: competitive exclusion, acid and bacteriocin production, immune modulation.
• Health benefits and products in market (probiotic curd, capsules).

Q5. Write notes on vinegar and sauerkraut production.

• Vinegar: alcoholic fermentation then acetic fermentation; Orleans (slow) and generator (quick) methods.
• Sauerkraut: shredded cabbage, salt, anaerobic, sequence of LAB.

Exam tip

Learn the organism for each fermented food in table form; "idli fermentation" and "probiotics vs prebiotics" are frequent short notes in Maharashtra universities.

Quick summary

Milk is a highly nutritious food and so a good medium for microbes. This topic covers the composition of milk, sources of contamination, types of milk spoilage, pasteurization and its tests, milk quality tests like the MBRT, and fermented dairy products such as curd, yoghurt, butter and cheese.

Key points

• Composition of cow milk (approximate): water about 87%, lactose about 4.5–5%, fat about 3.5–4%, proteins about 3.3% (mainly casein, plus whey proteins), minerals about 0.7%.
• Milk inside a healthy udder is nearly sterile, but it gets contaminated from the teat canal, udder skin, milker's hands, utensils, air, water and fodder.
• Normal milk flora: Lactococcus, Lactobacillus, Micrococcus, coliforms, yeasts and moulds.
• Milk-borne diseases: tuberculosis (Mycobacterium bovis), brucellosis (Brucella), Q fever (Coxiella burnetii), typhoid, streptococcal infections.
• Souring – lactic acid bacteria ferment lactose → lactic acid → curdling.
• Gassy fermentation – coliforms, Clostridium produce CO₂ and H₂.
• Proteolysis – protein breakdown, bitter taste (Bacillus, Pseudomonas).
• Lipolysis – fat breakdown → rancid odour.
• Ropy (slimy) milk – capsule-forming bacteria like Alcaligenes viscolactis.
• Colour changes – blue (Pseudomonas syncyanea), red (Serratia marcescens), yellow (Pseudomonas synxantha).
• Types of spoilage:
• Pasteurization: LTLT/holding method — 63 °C for 30 min; HTST — 72 °C for 15 s; UHT — about 135–150 °C for a few seconds (long shelf life in sealed packs).
• Coxiella burnetii is used as the index organism for pasteurization, as it is among the most heat-resistant non-spore-forming pathogens in milk.
• Phosphatase test: alkaline phosphatase is destroyed by proper pasteurization; its presence shows under-heating or mixing with raw milk.
• MBRT (methylene blue reduction test): methylene blue is decolourized by microbes as they use oxygen; faster decolourization = poorer quality.
• Other tests: standard plate count, coliform count, resazurin test, clot-on-boiling test.
• Fermented products: curd/dahi (Lactococcus lactis, Lactobacillus), yoghurt (Streptococcus thermophilus + Lactobacillus delbrueckii subsp. bulgaricus), cheese (rennet + starter culture; ripening with moulds like Penicillium roqueforti in blue cheese, P. camemberti in Camembert), butter and buttermilk, kefir.

Important terms

• Starter culture – selected microbes added to start a fermentation.
• Rennet – enzyme (chymosin) that coagulates milk protein in cheese making.
• Pasteurization – heat treatment that kills pathogens but not all microbes.
• Mastitis – infection of the udder (Staphylococcus aureus, Streptococcus agalactiae).
• Index organism – organism used to set a process standard.

Key facts

MBRT grading (common textbook scale):

(Exact time bands vary slightly between textbooks and standards.)

5 important questions

Q1. What is the phosphatase test?

A test for proper pasteurization. Alkaline phosphatase, a natural milk enzyme, is destroyed at pasteurization temperatures; if it is still active, the milk was under-pasteurized or mixed with raw milk.

Q2. What is ropy milk?

Milk that becomes slimy and forms threads because of capsule-producing bacteria such as Alcaligenes viscolactis.

Q3. Describe the types of spoilage of milk.

• Souring by lactic acid bacteria.
• Gassy fermentation by coliforms, clostridia.
• Proteolysis (bitter milk).
• Lipolysis (rancid milk).
• Ropiness.
• Abnormal colours (blue, red, yellow) with organisms.

Q4. Explain the methods of pasteurization and their testing.

• LTLT: 63 °C, 30 min.
• HTST: 72 °C, 15 s, followed by rapid cooling.
• UHT: very high temperature for seconds.
• Purpose: kill pathogens (index Coxiella burnetii), increase shelf life.
• Tests: phosphatase test, standard plate count, coliform test.

Q5. Describe the MBRT and its significance.

• 10 mL milk + 1 mL methylene blue solution in a sterile tube.
• Incubate at 37 °C in a water bath.
• Note time taken for the blue colour to disappear.
• Microbes use dissolved oxygen and reduce the dye.
• Shorter time → more microbes → poor milk.
• Quick, cheap test used at collection centres.

Exam tip

Remember the yoghurt pair (S. thermophilus + L. bulgaricus) and both pasteurization temperatures — they are asked often.

Quick summary

Soil is full of microorganisms — bacteria, actinomycetes, fungi, algae and protozoa — that keep it fertile by breaking down organic matter and recycling nutrients. This topic covers soil microflora, the rhizosphere, the nitrogen cycle, nitrogen fixation, phosphate solubilization, and biofertilizers used in Indian agriculture.

Key points

• Soil microbes: bacteria (most numerous), actinomycetes (e.g. Streptomyces, give the earthy smell due to geosmin), fungi, algae (including cyanobacteria) and protozoa.
• Rhizosphere: narrow zone of soil around plant roots, rich in microbes because roots release sugars and amino acids (root exudates). R:S ratio compares microbial numbers in rhizosphere and non-rhizosphere soil (usually more than 1).
• Nitrogen fixation: N₂ → NH₃.
• Ammonification: organic nitrogen (proteins) → NH₃ (many decomposers).
• Nitrification: NH₄⁺ → NO₂⁻ (Nitrosomonas) → NO₃⁻ (Nitrobacter).
• Denitrification: NO₃⁻ → N₂ (Pseudomonas, Paracoccus) under anaerobic conditions.
• Nitrogen cycle steps:
• Symbiotic: Rhizobium in root nodules of legumes (gram, soybean, groundnut); Frankia in non-legumes like Casuarina; Anabaena azollae in the water fern Azolla.
• Free-living (non-symbiotic): Azotobacter (aerobic), Clostridium (anaerobic), Beijerinckia, cyanobacteria like Nostoc and Anabaena.
• Associative: Azospirillum with roots of cereals and grasses.
• Biological nitrogen fixation uses the enzyme nitrogenase, which is damaged by oxygen and needs much ATP.
• Leghaemoglobin in nodules (pink colour) binds oxygen and protects nitrogenase.
• Phosphate-solubilizing microbes (PSM) like Bacillus megaterium, Pseudomonas, Aspergillus niger release organic acids that dissolve insoluble phosphates.
• Mycorrhiza: fungus–root partnership; VAM/AM fungi (e.g. Glomus) increase phosphorus and water uptake.
• Biofertilizers: preparations of living microbes that improve nutrient supply to plants. Types: nitrogen-fixing (Rhizobium, Azotobacter, Azospirillum, blue-green algae, Azolla), phosphate-solubilizing, potassium-mobilizing, mycorrhizal.
• Carrier: lignite or peat powder is commonly used to carry the microbes.
• Application: seed treatment (seed coated with slurry), seedling root dip (e.g. rice), soil application.
• Advantages: cheap, renewable, eco-friendly, improve soil health; reduce chemical fertilizer use. Limitations: slower action, short shelf life, sensitive to heat and chemicals, need specific strains for specific crops.
• Composting and vermicomposting use microbes (and earthworms) to turn organic waste into manure.

Important terms

• Rhizosphere – soil zone influenced by roots.
• Nitrogenase – enzyme complex that reduces N₂ to NH₃.
• Root nodule – swelling on legume roots where Rhizobium fixes nitrogen.
• Inoculant – biofertilizer culture applied to seed or soil.
• Humus – dark, stable organic matter formed by decomposition.

Formulas

• Nitrogen fixation (overall): N₂ + 8H⁺ + 8e⁻ + 16 ATP → 2NH₃ + H₂ + 16 ADP + 16 Pi
• Nitritation: 2NH₄⁺ + 3O₂ → 2NO₂⁻ + 4H⁺ + 2H₂O (Nitrosomonas)
• Nitratation: 2NO₂⁻ + O₂ → 2NO₃⁻ (Nitrobacter)
• Denitrification: NO₃⁻ → NO₂⁻ → NO → N₂O → N₂

5 important questions

Q1. What is the rhizosphere effect?

The increase in the number and activity of microorganisms in the soil around roots, because roots release nutrients (exudates). It is expressed as the R:S ratio.

Q2. Name two symbiotic and two free-living nitrogen fixers.

Symbiotic: Rhizobium (legumes), Frankia (Casuarina). Free-living: Azotobacter, Clostridium (also cyanobacteria like Nostoc).

Q3. Describe the nitrogen cycle with the role of microbes.

• Fixation: Rhizobium, Azotobacter, cyanobacteria.
• Ammonification: decomposers.
• Nitrification: Nitrosomonas, Nitrobacter.
• Assimilation by plants.
• Denitrification: Pseudomonas.
• Draw cycle diagram with organisms on arrows.

Q4. What are biofertilizers? Explain their types, application and advantages.

• Definition.
• Types: N₂-fixing, P-solubilizing, mycorrhizal, K-mobilizing, BGA/Azolla for paddy.
• Production: mass culture, mixing with carrier (lignite), packing.
• Application: seed treatment, root dip, soil application.
• Advantages and limitations.

Q5. Explain the formation of root nodules in legumes.

• Root releases flavonoids; Rhizobium makes Nod factors.
• Root hair curls around bacteria.
• Infection thread forms and carries bacteria into the cortex.
• Cortical cells divide → nodule.
• Bacteria become bacteroids; leghaemoglobin protects nitrogenase.
• NH₃ supplied to plant; plant gives carbohydrates.

Exam tip

Draw the nitrogen cycle with organism names written on each arrow; it fetches full marks in a 5-mark answer.

Quick summary

Plants suffer from diseases caused by fungi, bacteria, viruses, phytoplasmas and nematodes, and these cause huge crop losses in India every year. Plant pathology (phytopathology) studies the causes, symptoms, spread and control of these diseases. This topic covers how pathogens attack plants, the main symptoms, important bacterial, fungal and viral diseases of Maharashtra and Indian crops, and methods of control.

Key points

• Disease triangle: a disease needs a susceptible host, a virulent pathogen and a favourable environment (humidity, temperature, rain).
• Fungi cause most plant diseases (roughly 70–80%), then viruses, bacteria and others.
• How pathogens enter: directly through the cuticle (fungi with appressoria), through natural openings (stomata, hydathodes, lenticels) and through wounds (most bacteria).
• Weapons of pathogens: cell wall-degrading enzymes (pectinases, cellulases – cause soft rot), toxins (e.g. victorin, tabtoxin), growth regulators (auxin in crown gall), and blocking of xylem (wilts).
• Common symptoms: leaf spots, blights, wilts, rots, cankers, galls, mosaic, yellowing (chlorosis), stunting, rusts, smuts, mildews.
• Citrus canker – Xanthomonas citri; raised corky lesions on leaves and fruits (important for Nagpur oranges and Vidarbha citrus).
• Bacterial blight of rice – Xanthomonas oryzae pv. oryzae; leaf tips dry and turn yellow-white.
• Bacterial wilt of tomato, brinjal and potato – Ralstonia solanacearum; sudden wilting, bacterial ooze from cut stem.
• Crown gall – Agrobacterium tumefaciens; tumours from transfer of T-DNA of the Ti plasmid (used in plant genetic engineering).
• Soft rot of vegetables – Pectobacterium (formerly Erwinia) carotovorum.
• Bacterial diseases:
• Rust of wheat – Puccinia graminis tritici (black/stem rust); rust-coloured pustules.
• Late blight of potato – Phytophthora infestans (an oomycete, a fungus-like organism); caused the Irish famine of 1845–47.
• Blast of rice – Magnaporthe oryzae (Pyricularia oryzae).
• Wilt of cotton and pigeon pea (tur) – Fusarium spp.
• Powdery mildew (grapes, peas) and downy mildew (grapes, bajra).
• Red rot of sugarcane – Colletotrichum falcatum.
• Fungal diseases:
• Viral diseases: tobacco mosaic (TMV, mechanical spread), leaf curl of tomato and papaya ring spot (whitefly or aphid vectors), banana bunchy top (aphid), yellow vein mosaic of okra (whitefly).
• Phytoplasma diseases: grassy shoot of sugarcane, little leaf of brinjal, sandal spike (spread by leafhoppers).
• Cultural: crop rotation, resistant varieties, removal of infected plants, clean seed.
• Chemical: fungicides (Bordeaux mixture – copper sulphate + lime, discovered by Millardet in 1882; mancozeb, carbendazim), seed treatment, antibiotics like streptocycline for bacterial diseases.
• Biological: Trichoderma viride, Pseudomonas fluorescens, Bacillus subtilis.
• Legal: quarantine to stop entry of new pathogens.
• Integrated disease management (IDM) combines all.
• Control methods:

Important terms

• Pathogen – organism that causes disease in a plant.
• Symptom – visible change in a diseased plant (spot, wilt).
• Appressorium – flattened tip of a fungal germ tube that presses into the host surface.
• Vector – organism (insect) that carries a pathogen from plant to plant.
• Bordeaux mixture – copper sulphate + lime fungicide.

Key facts

5 important questions

Q1. What is the disease triangle?

It shows that a plant disease develops only when three factors meet: a susceptible host, a virulent pathogen and a favourable environment. Removing any one prevents the disease.

Q2. Name the causal organism and one symptom of citrus canker.

Xanthomonas citri (Gram-negative rod). It forms raised, brown, corky lesions with a yellow halo on leaves, twigs and fruits.

Q3. Describe the mechanisms by which pathogens attack plants.

• Entry: direct, natural openings, wounds.
• Enzymes degrading cuticle and cell wall.
• Toxins and growth regulators.
• Vascular blocking in wilts.
• Defence of plants (brief).

Q4. Describe two bacterial diseases of plants with symptoms and control.

• Citrus canker and bacterial blight of rice (or crown gall).
• Pathogen, symptoms, spread, control (copper sprays, streptocycline, resistant varieties, pruning).

Q5. Explain the methods of controlling plant diseases.

• Cultural, chemical, biological, legal (quarantine), host resistance.
• Integrated disease management.
• Examples from Maharashtra crops.

Exam tip

Learn one bacterial, one fungal and one viral disease fully (pathogen, symptoms, control). Crown gall links to genetic engineering — mention the Ti plasmid.

Quick summary

Water can carry disease-causing microbes, so testing and purifying drinking water is a key public-health job. This topic covers water-borne diseases, indicator organisms, bacteriological tests of water (MPN and membrane filter), water purification, and how sewage is treated by primary, secondary and tertiary methods.

Key points

• Water-borne diseases: cholera (Vibrio cholerae), typhoid (Salmonella Typhi), bacillary dysentery (Shigella), hepatitis A and E (viruses), polio, amoebic dysentery (Entamoeba histolytica), giardiasis (Giardia).
• Testing for every pathogen is not practical, so we test for indicator organisms that show faecal pollution.
• Coliforms (Gram-negative, non-spore-forming rods that ferment lactose with acid and gas within 48 h at 35–37 °C) are the usual indicators. Faecal (thermotolerant) coliforms, especially E. coli, grow at 44.5 °C and show recent faecal contamination.
• Other indicators: faecal streptococci (enterococci), Clostridium perfringens (shows older or intermittent pollution, as spores survive long).
• An ideal indicator: present in faeces in large numbers, absent from clean water, survives slightly longer than pathogens, easy and quick to detect, does not multiply in water.
• Presumptive test: water inoculated into lactose broth (or MacConkey broth) tubes with Durham's tubes; acid and gas = positive.
• Confirmed test: positive tubes subcultured onto EMB or Endo agar (or BGLB broth); E. coli gives colonies with a green metallic sheen on EMB.
• Completed test: colonies checked by Gram stain (Gram-negative rods) and gas in lactose broth.
• MPN (multiple tube fermentation) test — three stages:
• The MPN (most probable number) of coliforms per 100 mL is read from statistical MPN tables.
• Membrane filter technique: known volume (e.g. 100 mL) filtered through a 0.45 µm membrane; membrane placed on selective medium (e.g. m-Endo); colonies counted directly. Fast and precise.
• IMViC tests (Indole, Methyl red, Voges–Proskauer, Citrate) separate E. coli (+ + − −) from Enterobacter aerogenes (− − + +).
• Water purification (municipal): sedimentation → coagulation/flocculation (alum) → filtration (slow or rapid sand filters) → disinfection (chlorination, ozone, UV).
• Primary: screening, grit removal, sedimentation — removes solids.
• Secondary (biological): aerobic microbes oxidize organic matter — activated sludge process, trickling filter, oxidation ponds; anaerobic digestion of sludge produces biogas (mainly methane and CO₂).
• Tertiary: removes remaining nitrogen, phosphorus, and pathogens (chemical precipitation, filtration, chlorination).
• Sewage treatment:
• BOD (biochemical oxygen demand): oxygen used by microbes to oxidize organic matter in water in 5 days at 20 °C (BOD₅). High BOD = more pollution. COD uses a chemical oxidant (dichromate) and is usually higher than BOD.

Important terms

• Indicator organism – organism whose presence shows faecal pollution.
• Potable water – water safe to drink.
• Activated sludge – flocs of microbes recycled in aeration tanks.
• Residual chlorine – chlorine remaining after disinfection that keeps protecting water.
• Eutrophication – excess nutrients causing algal blooms and oxygen loss.

Formulas

• D₁ = dissolved oxygen (DO) of diluted sample at start (mg/L)
• D₂ = DO after 5 days at 20 °C (mg/L)
• P = decimal fraction of sample used in the dilution
• BOD (mg/L) = (D₁ − D₂) ÷ P
• Example: D₁ = 8.0 mg/L, D₂ = 3.5 mg/L, P = 0.02 (2% sample) → BOD = (8.0 − 3.5) ÷ 0.02 = 4.5 ÷ 0.02 = 225 mg/L.

5 important questions

Q1. Why is E. coli used as an indicator of faecal pollution?

It lives only in the intestine of humans and warm-blooded animals, is passed in large numbers in faeces, does not normally multiply in clean water and is easy to detect. So its presence shows recent faecal contamination.

Q2. Define BOD.

Biochemical oxygen demand — the amount of dissolved oxygen used by microorganisms to break down organic matter in a water sample during 5 days at 20 °C. It measures organic pollution.

Q3. Describe the MPN test for coliforms.

• Presumptive: lactose broth tubes with Durham's tubes (e.g. 3 × 10 mL, 3 × 1 mL, 3 × 0.1 mL sample), 35–37 °C, 24–48 h; acid + gas = positive.
• Confirmed: streak on EMB/Endo; typical colonies (metallic sheen).
• Completed: Gram stain, lactose fermentation.
• Read MPN/100 mL from tables.
• IMViC to identify E. coli.

Q4. Explain secondary treatment of sewage.

• Biological oxidation of organic matter by aerobic microbes.
• Activated sludge: aeration tank, flocs of bacteria and protozoa, settling, sludge recycled.
• Trickling filter: sewage sprinkled over stones covered with microbial film.
• Oxidation ponds: algae–bacteria partnership.
• Sludge digestion: anaerobic, produces methane-rich biogas.
• Lowers BOD greatly.

Q5. Calculate BOD: D₁ = 7.8 mg/L, D₂ = 4.2 mg/L, 5 mL sample diluted to 300 mL.

• P = 5/300 = 0.0167
• D₁ − D₂ = 7.8 − 4.2 = 3.6 mg/L
• BOD = 3.6 ÷ 0.0167 ≈ 216 mg/L (exactly 3.6 × 60 = 216 mg/L)
• Indicates a heavily polluted sample.

Exam tip

Draw the three stages of the MPN test as a flowchart, and the sewage treatment plant as a block diagram (primary → secondary → tertiary).

Quick summary

Industrial microbiology uses microbes on a large scale to make useful products — alcohol, antibiotics, organic acids, enzymes, vitamins and vaccines. This topic covers the fermenter (bioreactor) and its parts, types of fermentation, strain improvement, media, downstream processing, and production of key products like penicillin, ethanol and citric acid.

Key points

• In industry, "fermentation" means any large-scale culture of microbes to make a product, aerobic or anaerobic.
• Primary metabolites are made during the growth (log) phase and are essential for growth (ethanol, amino acids, organic acids). Secondary metabolites are made mostly in the stationary phase (antibiotics, pigments).
• Fermenter (bioreactor) — usually stainless steel. Parts: vessel, impeller/agitator (mixing), sparger (air bubbles), baffles (prevent vortex), cooling jacket or coils, probes for pH, temperature, dissolved oxygen and foam, inlet/outlet ports, sampling port.
• Types of fermenters: stirred tank (most common), airlift, bubble column, packed bed.
• Batch, fed-batch (nutrients added gradually, e.g. penicillin, baker's yeast) and continuous fermentation.
• Submerged (liquid) and solid-state fermentation (on moist solids like wheat bran, e.g. some enzymes).
• Upstream processing: strain selection and improvement (mutation, recombination, genetic engineering), media formulation, sterilization, inoculum development.
• Industrial media use cheap raw materials: molasses (sugar), corn steep liquor (nitrogen, used in penicillin production), whey, starch.
• Downstream processing: separation of cells (filtration, centrifugation), cell disruption (if product is inside), extraction, purification (precipitation, chromatography), concentration, drying, packaging.
• Antifoam agents (silicone oils, vegetable oils) control foam.
• Penicillin: Penicillium chrysogenum (high-yield strains); fed-batch, aerobic, 25–26 °C; precursor phenylacetic acid added to get penicillin G; corn steep liquor in medium.
• Ethanol: Saccharomyces cerevisiae on molasses (sugarcane industry, important in Maharashtra); anaerobic; product distilled. Zymomonas mobilis is another producer.
• Citric acid: Aspergillus niger, aerobic, on molasses or sucrose, with low iron/manganese and low pH.
• Other products: vinegar/acetic acid (Acetobacter aceti), lactic acid (Lactobacillus delbrueckii), glutamic acid (Corynebacterium glutamicum), vitamin B₁₂ (Propionibacterium, Pseudomonas denitrificans), amylase and protease (Bacillus, Aspergillus), streptomycin (Streptomyces griseus).

Important terms

• Bioreactor – vessel for growing microbes under controlled conditions.
• Inoculum – starter culture added to the fermenter.
• Precursor – compound added that becomes part of the product.
• Downstream processing – recovery and purification of product.
• Scale-up – moving a process from flask to pilot to industrial size.

Key facts

Equations:

• Ethanol: C₆H₁₂O₆ → 2C₂H₅OH + 2CO₂
• Vinegar: C₂H₅OH + O₂ → CH₃COOH + H₂O

5 important questions

Q1. Differentiate primary and secondary metabolites.

Primary metabolites are formed during active growth and are essential for the cell (ethanol, amino acids). Secondary metabolites are formed mainly in the stationary phase and are not essential for growth (antibiotics, toxins).

Q2. What is the role of the sparger and baffles in a fermenter?

The sparger releases sterile air as fine bubbles for aeration. Baffles are metal strips on the wall that break the vortex and improve mixing.

Q3. Describe the design of a typical stirred-tank fermenter.

• Vessel of stainless steel with headspace.
• Agitator and impellers; motor.
• Sparger; air filter.
• Baffles.
• Cooling jacket/coils.
• Probes: pH, temperature, DO, foam.
• Ports: inoculation, sampling, harvest, acid/base and antifoam addition.
• Draw and label.

Q4. Explain industrial production of penicillin.

• Strain: high-yield P. chrysogenum.
• Inoculum: spores → seed tanks.
• Medium: corn steep liquor, lactose/glucose feed, phenylacetic acid.
• Fed-batch, aerobic, pH about 6.5, 25–26 °C, several days.
• Recovery: filter mycelium, solvent extraction at low pH, back extraction, crystallization as sodium/potassium salt.
• Semi-synthetic penicillins from 6-APA.

Q5. Describe production of ethanol by fermentation.

• Raw material: molasses diluted, pH adjusted (about 4–5), nutrients added.
• Yeast inoculum (S. cerevisiae).
• Anaerobic fermentation at about 30 °C for 2–3 days.
• Distillation gives rectified spirit (about 95%); further dehydration for absolute alcohol/fuel ethanol.
• By-products: CO₂, fusel oils.
• Uses: fuel blending, solvents, beverages, pharmaceuticals.

Exam tip

Learn product – organism – key condition as a table, and practise drawing the fermenter with at least 10 labels.

Quick summary

Besides antibiotics and alcohol, microorganisms are used to manufacture enzymes, amino acids and vitamins on an industrial scale. Microbes are preferred because they grow fast on cheap media, can be improved genetically and give products in large amounts. This topic covers the production of amylase and protease, glutamic acid and lysine, and vitamin B₁₂ and riboflavin, along with strain improvement.

Key points

• Why microbial enzymes? Fast growth, easy scale-up, no seasonal dependence, easy genetic improvement, and many are extracellular (secreted into the medium), making recovery simple.
• α-Amylase – Bacillus amyloliquefaciens, B. licheniformis (heat-stable, used in starch liquefaction), Aspergillus oryzae.
• Proteases – Bacillus licheniformis (subtilisin; alkaline protease used in detergents), Aspergillus.
• Glucose isomerase – Streptomyces spp.; converts glucose to fructose for high-fructose syrup.
• Pectinase – Aspergillus niger; clears fruit juices.
• Lipase, cellulase (Trichoderma reesei), rennet substitute (microbial chymosin made in recombinant microbes).
• Main industrial enzymes and producers:
• Production steps for an enzyme: strain selection → medium (starch, corn steep liquor, soybean meal) → submerged or solid-state fermentation (wheat bran for fungal enzymes) → remove cells (filtration/centrifugation) → concentrate (ultrafiltration) → precipitate (ammonium sulphate or alcohol) → dry or formulate → immobilization for repeated use.
• L-Glutamic acid (sold as monosodium glutamate, MSG) – Corynebacterium glutamicum (discovered by Kinoshita, Japan, 1957).
• Key trick: biotin limitation (or adding penicillin or surfactants) makes the membrane leaky so glutamate is excreted.
• Aerobic, pH about 7–8, 30–37 °C; ammonium supplied as nitrogen source.
• L-Lysine – auxotrophic or regulatory mutants of C. glutamicum (Brevibacterium flavum) that escape feedback inhibition; used in animal feed.
• Amino acid production:
• Vitamin B₁₂ (cyanocobalamin) – made only by microbes; Pseudomonas denitrificans and Propionibacterium shermanii; needs cobalt and DMB (5,6-dimethylbenzimidazole); cyanide added during recovery converts it to cyanocobalamin.
• Riboflavin (vitamin B₂) – fungi Ashbya gossypii and Eremothecium ashbyii, and Bacillus subtilis.
• Vitamin C – partly microbial (Reichstein process step using Gluconobacter, or two-step fermentation).
• Vitamin production:
• Regulation tricks: use of auxotrophic mutants and feedback-resistant mutants so that cells overproduce the product.
• Strain improvement methods: mutation (UV, chemicals) and screening, protoplast fusion, recombinant DNA, site-directed mutagenesis.
• Uses: detergents (proteases, lipases), food (amylase in baking, pectinase in juice), textiles (amylase for desizing), leather (protease for dehairing), medicine (streptokinase, asparaginase), feed (lysine), flavour (MSG).

Important terms

• Extracellular enzyme – enzyme secreted out of the cell.
• Immobilized enzyme – enzyme fixed on a support so it can be reused.
• Auxotroph – mutant that cannot make a nutrient and needs it in the medium.
• Feedback inhibition – end product inhibits the first enzyme of its pathway.
• Solid-state fermentation – growth of microbes on moist solid substrate with little free water.

Key facts

5 important questions

Q1. Why is biotin kept limited in glutamic acid fermentation?

Biotin is needed for fatty acid synthesis. Under biotin limitation the cell membrane becomes weak and leaky, so glutamic acid leaks out of the cell into the medium instead of being used inside, giving a high yield.

Q2. Name two microorganisms used for vitamin B₁₂ production.

Pseudomonas denitrificans and Propionibacterium shermanii (P. freudenreichii). Cobalt must be added to the medium.

Q3. Describe the industrial production of amylase.

• Organism and its features.
• Medium, fermentation conditions (submerged or solid-state).
• Downstream recovery and formulation.
• Applications in starch, baking, textile and brewing industries.

Q4. Explain the production of L-glutamic acid by fermentation.

• Corynebacterium glutamicum; medium (glucose/molasses, ammonia, minerals, limiting biotin).
• Conditions: aerobic, pH, temperature, duration about 2–3 days.
• Mechanism of excretion.
• Recovery as MSG by crystallization.
• Flow chart.

Q5. Write a note on microbial production of vitamins.

• B₁₂: organisms, cobalt, DMB, recovery as cyanocobalamin.
• Riboflavin: Ashbya gossypii, flavinogenic fungi.
• Uses and importance.

Exam tip

Remember organism–product pairs from the table; examiners often ask "name the organism used for …". Draw one simple flow chart for any production process.

Quick summary

Single cell protein (SCP) is the dried cells (biomass) of microorganisms such as algae, yeasts, fungi and bacteria, used as a protein source for humans or animals. Mushrooms are edible fleshy fruiting bodies of certain fungi grown on agricultural waste. Both turn cheap or waste materials into protein-rich food, which matters for a country like India with protein malnutrition and plenty of crop residue.

Key points

• The term single cell protein was coined by Carroll L. Wilson (MIT) in 1966.
• Advantages of SCP organisms: very fast growth (bacteria double in 20–120 min, yeasts in about 1–3 h), high protein content, grow on cheap or waste substrates, need little land and water, not affected by season, and can be genetically improved.
• Protein content (dry weight, approximate): bacteria 50–80%, yeasts 45–55%, algae 40–60%, moulds 30–50%.
• Algae: Spirulina (cyanobacterium; about 60–70% protein, grown in alkaline ponds), Chlorella, Scenedesmus.
• Yeasts: Saccharomyces cerevisiae, Candida utilis (grows on sulphite waste liquor, molasses), Kluyveromyces on whey.
• Fungi: Fusarium venenatum (Quorn mycoprotein, UK), Trichoderma, Chaetomium on cellulose wastes.
• Bacteria: Methylophilus methylotrophus on methanol (ICI's "Pruteen", animal feed); Pseudomonas on hydrocarbons.
• Organisms and substrates:
• Production steps: substrate preparation → sterilization → fermentation (continuous, aerated) → harvesting (filtration/centrifugation) → washing → reduction of nucleic acids → drying.
• Problems / limitations: high nucleic acid content (bacteria up to about 15%) — excess raises uric acid and can cause gout and kidney stones (reduced by heat treatment that activates the cells' own RNases); possible toxins or contaminants; poor taste and digestibility of cell walls; high cost; public acceptance.
• Mushroom biology: mushrooms are mostly Basidiomycetes. The fruiting body has a stalk (stipe), cap (pileus) and gills bearing basidiospores. The underground mycelium is the vegetative part.
• Button mushroom – Agaricus bisporus; temperate (about 14–18 °C for fruiting); grown on composted straw with a casing layer. Main producer areas include Himachal Pradesh and Haryana; also grown in climate-controlled units.
• Oyster mushroom (dhingri) – Pleurotus spp.; grows at about 20–30 °C; easiest for Maharashtra farmers; grown on chopped, soaked and pasteurized paddy or wheat straw in polythene bags.
• Paddy straw mushroom – Volvariella volvacea; tropical, 30–35 °C.
• Milky mushroom – Calocybe indica; suited to hot regions.
• Common cultivated mushrooms in India:
• Steps of oyster mushroom cultivation: spawn preparation (mycelium on boiled wheat/sorghum grains) → substrate (straw) chopped, soaked and pasteurized (hot water about 80 °C or chemical) → spawning (layering spawn in bags, about 2–5% of wet substrate weight) → spawn run in dark (about 2–3 weeks, white mycelium covers straw) → bags cut, high humidity (80–90%), light and ventilation → cropping of fruiting bodies in flushes.
• Nutritional value: mushrooms are rich in protein (about 20–35% dry weight), B vitamins, minerals and fibre, and low in fat; they are good for diabetics and heart patients. Wild mushrooms can be poisonous (e.g. Amanita phalloides).
• ICAR–Directorate of Mushroom Research, Solan (Himachal Pradesh), leads mushroom research in India.

Important terms

• Single cell protein – dried microbial biomass used as protein food or feed.
• Mycoprotein – protein-rich food made from fungal mycelium.
• Spawn – pure mushroom mycelium grown on grain, used as "seed".
• Spawn run – period when mycelium spreads through the substrate.
• Casing – layer of soil-like material placed on compost to induce fruiting of button mushrooms.

Key facts

5 important questions

Q1. Why is the high nucleic acid content of SCP a problem?

Humans break nucleic acids down to uric acid. Too much uric acid in blood can cause gout and kidney stones, so the RNA content of SCP for human food must be lowered.

Q2. What is spawn?

Spawn is the pure culture of mushroom mycelium grown on sterilized cereal grains (wheat, sorghum). It is used like seed to inoculate the substrate.

Q3. What is SCP? Give its advantages and limitations.

• Definition and term origin.
• Organisms with substrates (table).
• Advantages: fast growth, high protein, waste use, no land.
• Limitations: nucleic acids, toxins, digestibility, cost, acceptance.

Q4. Describe the production of SCP from algae or yeast.

• Spirulina: open raceway ponds, alkaline medium (sodium bicarbonate), harvesting by filtration, drying.
• Or yeast on molasses/whey: continuous aerated fermentation, centrifugation, RNA reduction, drying.
• Uses as food supplement and feed.

Q5. Explain the cultivation of oyster mushroom.

• Spawn preparation.
• Substrate preparation and pasteurization.
• Spawning and bagging.
• Spawn run and cropping conditions.
• Yield, harvesting and nutritional value.

Exam tip

Write the cultivation steps as a numbered flow chart. For SCP, always mention the nucleic acid problem — it is the most common short question.

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.