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TU B.Sc. First Year Syllabus: Microbiology
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TU B.Sc. First Year Microbiology (MB 101) — Complete Syllabus Overview

Microbiology is one of the major subjects offered under the Four-Year Bachelor of Science (B.Sc.) programme of Tribhuvan University’s Institute of Science and Technology (IoST), Kirtipur, Kathmandu. The First Year Microbiology course, officially titled General Microbiology (Course No. MB 101), is a 100-mark theory paper accompanied by a 50-mark practical paper (MB 102), making the combined First Year Microbiology assessment worth 150 marks. The current syllabus is the Revised Course of Study 2073 BS (December 2016), developed by the IoST Microbiology Subject Committee.

The course spans 150 lecture hours and covers the full breadth of introductory microbiology: the history and scope of the discipline, classification and nomenclature of microorganisms, detailed bacteriology (morphology, physiology, growth, metabolism, and genetics), microscopy and laboratory techniques, sterilization and disinfection, host-parasite interactions and immunity, and introductions to virology, parasitology, and mycology. It is the foundational course for all four years of the B.Sc. Microbiology programme.

Subject Information at a Glance

  • Course Title: General Microbiology
  • Course No.: MB 101 (Theory, Major) | MB 102 (Practical)
  • Year: First Year — Four Year B.Sc. Microbiology Programme
  • Full Marks: 100 (Theory) + 50 (Practical) = 150 total
  • Pass Marks: 35 (Theory) | 20 (Practical) — both must be passed separately
  • Total Lecture Hours: 150 (Theory)
  • Examination: Annual (end-of-year exam conducted by IoST, TU)
  • Conducting Institution: Institute of Science and Technology (IoST), Tribhuvan University
  • Curriculum Version: Revised Course of Study 2073 BS (December 2016)
  • Eligibility: Passed +2 Science (Biological Stream) or equivalent with minimum C grade in all subjects; must pass the IoST entrance examination
  • Total Seats: Approximately 700 students per year across 25 TU colleges (8 constituent + 17 affiliated)

Why Microbiology?

Microbiology — the scientific study of microorganisms too small to see with the naked eye — is one of the most dynamic and impactful disciplines in modern biological science. Bacteria, viruses, fungi, and parasites collectively shape human health, agriculture, food systems, ecosystems, and industrial production in ways that touch every aspect of daily life. From the development of antibiotics to the engineering of vaccines, from food fermentation to bioremediation of polluted environments, microbiologists are at the forefront of addressing humanity’s most pressing challenges.

In Nepal’s context, microbiology has particular urgency. Infectious diseases — including tuberculosis, typhoid, cholera, hepatitis, HIV/AIDS, and antimicrobial-resistant bacterial infections — remain among the leading causes of morbidity and mortality, particularly in rural and peri-urban communities. Nepal’s expanding healthcare infrastructure, growing pharmaceutical sector, food processing industry, and environmental monitoring agencies all require trained microbiologists. At TU, the four-year B.Sc. Microbiology programme is explicitly designed to cover medical microbiology, environmental and public health microbiology, food microbiology, agricultural microbiology, and microbial biotechnology — four distinct applied domains taught as an integrated discipline, with specialization possible at the M.Sc. level.

MB 101 Theory Syllabus — Topic-wise Detail

Topic 1: History and Development of Microbiology (4 teaching hours)

  • Development of microbiology with reference to the modern era: key historical milestones from ancient observations of fermentation and disease through to the establishment of microbiology as a formal scientific discipline
  • Important discoveries in the history of microbiology: van Leeuwenhoek’s invention of the microscope and first observation of “animalcules”; discovery of oxygen (Priestley, Scheele); Jenner’s smallpox vaccination (1796); Semmelweis and handwashing; Lister’s antiseptic surgery
  • Theory of spontaneous generation: historical background; key experiments arguing for and against — van Helmont’s recipe, Needham’s broth experiments (supporting), Spallanzani’s sealed flasks (refuting)
  • Louis Pasteur’s contributions: swan-neck flask experiment (definitive refutation of spontaneous generation); germ theory of fermentation; pasteurization; development of vaccines for chicken cholera, anthrax, and rabies; role of yeast in alcoholic fermentation
  • Robert Koch’s contributions: isolation of Bacillus anthracis (1876), Mycobacterium tuberculosis (1882), Vibrio cholerae (1883); development of pure culture techniques (solid media using gelatin, then agar — suggested by Angelina Fanny Hesse); Koch’s postulates — definition, steps, limitations; Nobel Prize in Physiology or Medicine 1905
  • Establishment of germ theory of disease: the shift from miasma theory to germ theory; implications for medicine, surgery, and public health

Topic 2: Classification and Nomenclature of Microorganisms (15 teaching hours)

  • Classification schemes of living organisms: purpose of biological classification (communication, identification, predictive value); artificial vs. natural vs. phylogenetic classification
  • Differential characteristics of prokaryotic and eukaryotic microorganisms: cell wall, membrane, nucleus, ribosomes, organelles, DNA organization, reproduction — tabular comparison
  • General principles of nomenclature: binomial nomenclature (Linnaean system); rules of the International Code of Nomenclature of Bacteria (ICNB); type cultures; valid publication; formal vs. informal names
  • Three-domain concept (Woese and Fox 1990): Bacteria, Archaea, and Eukarya — basis in 16S/18S rRNA sequence comparison; distinguishing features of each domain
  • Five-kingdom system (Whittaker 1969): Monera, Protista, Fungi, Plantae, Animalia — basis, placement of microorganisms in each kingdom; limitations
  • Basic understanding of classification of bacteria: traditional phenotypic classification (morphology, Gram staining, spore formation, oxygen requirements, motility) vs. modern molecular classification (16S rRNA gene sequencing, DNA-DNA hybridization, GC content)
  • Bergey’s Manual of Systematic Bacteriology: history, editions (1st edition 1923; current editions organized by molecular phylogeny); role as the primary reference for bacterial taxonomy; organization of bacterial groups (proteobacteria, firmicutes, bacteroidetes, actinobacteria — overview)
  • Classification of viruses: Baltimore classification system (Groups I–VII based on genome type and replication strategy); ICTV classification (realm, kingdom, phylum, class, order, family, genus, species)
  • Classification of fungi: major groups — Chytridiomycota, Zygomycota, Ascomycota, Basidiomycota, Deuteromycota (Fungi imperfecti); basis of classification (sexual reproduction, spore types)
  • Classification of protozoa: basis of classification (locomotion organelle — flagella, cilia, pseudopods, apical complex); major groups — Mastigophora, Sarcodina, Ciliophora, Apicomplexa; medically important genera (Giardia, Entamoeba, Paramecium concept, Plasmodium, Toxoplasma, Cryptosporidium)

Topic 3: Scope and Applications of Microbiology (7 teaching hours)

  • Harmful microorganisms: infectious diseases in humans, animals, and plants; food spoilage and foodborne illness; biodeterioration of materials
  • Beneficial microorganisms: decomposition and nutrient cycling; soil fertility (nitrogen-fixing bacteria — Rhizobium, Azotobacter; phosphate solubilizers); symbiotic gut microbiota
  • Applied microbiology — major branches and their applications:
    • Medical microbiology: diagnosis, treatment, and prevention of infectious diseases; antibiotic discovery and resistance; vaccine development
    • Public health microbiology: epidemiology of communicable diseases, surveillance, water and food safety monitoring; relevant to Nepal — tuberculosis (highest burden in South Asia), typhoid, cholera, HIV, hepatitis B
    • Agricultural microbiology: biofertilizers, biopesticides, plant disease diagnosis, mycorrhizal associations, rhizobium inoculants; relevance to Nepal’s subsistence agriculture
    • Food microbiology: fermented foods (yoghurt, cheese, gundruk, sinki, tongba, chaang — Nepal-specific fermented foods), food spoilage prevention, HACCP principles
    • Microbial biotechnology: recombinant DNA technology, production of insulin, growth hormone, erythropoietin, interferon using engineered microbes; gene cloning; PCR
    • Environmental microbiology: bioremediation of oil spills and heavy metal contamination; sewage treatment (aerobic and anaerobic processes); microorganisms in the carbon and nitrogen cycles
    • Industrial microbiology: production of antibiotics (penicillin, streptomycin, tetracycline), vitamins, amino acids, organic acids, enzymes, and alcoholic beverages
    • Pharmaceutical microbiology: quality control in drug manufacturing; testing for microbial contamination; production of biologics

Topic 4: Morphology of Bacteria (10 teaching hours)

  • Structure of bacteria — overview: size (range 0.2–10 μm); prokaryotic nature
  • Shapes and arrangements: cocci (spherical — diplococci, tetracocci, streptococci, staphylococci, sarcinae); bacilli (rod-shaped — single, diplobacilli, streptobacilli, palisade arrangement); spirilla (curved rods — vibrio, spirillum, spirochete); other forms (filamentous, star-shaped, square)
  • Morphology and fine structure of cell organelles:
    • Cell wall: composition of peptidoglycan (murein) — glycan backbone (NAG-NAM) and peptide cross-links; differences in Gram-positive cell wall (thick peptidoglycan, teichoic acids) vs. Gram-negative cell wall (thin peptidoglycan, outer membrane with lipopolysaccharide — LPS, lipid A, core, O-antigen); role of LPS in endotoxin activity; acid-fast cell wall (Mycobacterium — mycolic acids)
    • Cell membrane: fluid mosaic model; functions (transport, energy generation, enzyme attachment); mesosome (concept and controversy)
    • Capsule and slime layer: composition (polysaccharide, polypeptide in Bacillus anthracis); functions — protection from phagocytosis, desiccation resistance, adhesion; virulence factor; capsule staining (negative staining)
    • Flagella: structure (filament, hook, basal body — L, P, S, M rings in Gram-negative; two rings in Gram-positive); arrangement (monotrichous, lophotrichous, amphitrichous, peritrichous); mechanism of rotation (proton motive force); chemotaxis
    • Pili and fimbriae: structure (pilin protein, hollow tube); types — common fimbriae (adhesion), sex pili (F pilus — DNA transfer in conjugation), type IV pili (twitching motility)
    • Ribosomes: 70S = 50S + 30S subunits; role in translation; target of antibiotics (chloramphenicol, erythromycin — 50S; streptomycin, tetracycline — 30S)
    • Inclusion bodies: types — metachromatic granules (volutin, polyphosphate), glycogen granules, PHB (polyhydroxybutyrate), sulfur globules, gas vesicles, carboxysomes — composition and function
    • Nucleoid: structure (single circular chromosome, supercoiled by gyrase, associated with HU proteins); compared to eukaryotic nucleus; plasmids
    • Endospores: formation (sporulation — key genera: Bacillus, Clostridium); structure (exosporium, outer coat, inner coat, cortex, germ cell wall, inner membrane, core); chemical composition (dipicolinic acid, SASPs); resistance to heat, radiation, desiccation, chemicals; significance in sterilization validation; germination
  • Differences between Gram-positive and Gram-negative bacteria: comprehensive comparison table — cell wall thickness, outer membrane, LPS, teichoic acids, periplasmic space, sensitivity to antibiotics, lysozyme, and detergents

Topic 5: Growth and Physiology of Bacteria (8 teaching hours)

  • Nutritional types of bacteria: classification by energy source (light — phototrophs; chemical — chemotrophs) and carbon source (CO₂ — autotrophs/lithotrophs; organic compounds — heterotrophs/organotrophs):
    • Photolithoautotrophs: cyanobacteria, purple sulfur bacteria
    • Photoorganoheterotrophs: purple non-sulfur bacteria
    • Chemolithoautotrophs: nitrifying bacteria (Nitrosomonas, Nitrobacter), sulfur-oxidizing bacteria (Thiobacillus), iron bacteria
    • Chemoorganoheterotrophs: most bacteria of medical importance
  • Nutritional requirements: macronutrients (C, H, O, N, S, P, K, Mg, Fe), micronutrients (trace elements — Mn, Mo, Zn, Cu, Co, Ni), growth factors (vitamins, amino acids, purines, pyrimidines)
  • Entry of nutrients: passive diffusion (small nonpolar molecules), facilitated diffusion (permease — no ATP), active transport (ATP-dependent — group translocation — phosphoenolpyruvate–phosphotransferase system, PTS, in bacteria)
  • Bacterial growth: binary fission — cell elongation, DNA replication, septum formation, cell separation; generation time (doubling time); calculation of generation time (g = t/n; N = N₀ × 2ⁿ)
  • Bacterial growth curve: four phases — lag phase (metabolic activity, enzyme synthesis, no cell division), exponential/log phase (maximum growth rate, metabolic activity, antibiotic sensitivity), stationary phase (growth = death, nutrient depletion, waste accumulation), death/decline phase (death > growth, spore formation in some); mathematical description; significance of each phase
  • Factors affecting bacterial growth:
    • Temperature: minimum, optimum, maximum; psychrophiles (0–20°C), mesophiles (20–45°C), thermophiles (45–80°C), hyperthermophiles (>80°C); psychrotrophs; clinical significance
    • pH: acidophiles, neutrophiles, alkaliphiles; internal pH buffering mechanisms; pH and food preservation
    • Osmotic pressure: halophiles (obligate and facultative), osmophiles; plasmolysis and plasmoptysis
    • Oxygen requirement: obligate aerobes, obligate anaerobes (toxic oxygen species — superoxide O₂⁻, hydrogen peroxide H₂O₂, hydroxyl radical OH•; lack of superoxide dismutase and catalase), facultative anaerobes, microaerophiles, aerotolerant anaerobes; oxygen-reducing enzymes (superoxide dismutase, catalase, peroxidase)

Topic 6: Isolation, Enumeration, and Culture of Bacteria (10 teaching hours)

  • Types of bacteriological culture media:
    • By physical state: liquid (broth), semi-solid (0.5% agar), solid (1.5–2% agar)
    • By composition: simple/basal media (nutrient broth, nutrient agar), complex/undefined media, chemically defined/synthetic media
    • By function: general purpose media, enrichment media (increase relative proportion of target organism — selenite F broth for Salmonella), selective media (inhibit unwanted organisms — MacConkey agar, TCBS agar, Mannitol salt agar), differential media (differentiate organisms by colour/reaction — blood agar, EMB agar, CLED agar), transport media (Cary-Blair, Stuart’s medium), special media (serum agar, chocolate agar, Löwenstein-Jensen for TB)
  • Techniques for isolation and enumeration of bacteria:
    • Streak plate technique: quadrant streaking, T-streaking — principle, procedure, limitations; obtaining pure colonies
    • Pour plate technique: serial dilution + agar pour; colonies grow within and on surface; total viable count
    • Spread plate technique: serial dilution + surface spreading; surface colonies only; preferred for enumeration
    • Membrane filtration technique: filter pore size 0.45 μm; used for water samples (coliform detection); filter placed on selective medium
    • Most Probable Number (MPN) method: statistical estimate of coliform count; 3-tube or 5-tube series; presumptive, confirmed, and completed tests; used for water quality testing
    • Direct microscopic count: Petroff-Hausser counting chamber for bacteria; Haemocytometer for larger cells; does not distinguish viable from dead cells; Breed smear method
  • Biochemical tests of bacteria: purpose (identification); commonly used tests — catalase test, oxidase test, indole test (IMViC series — Indole, Methyl Red, Voges-Proskauer, Citrate utilization), urease test, coagulase test, sugar fermentation tests, triple sugar iron (TSI) agar; MALDI-TOF mass spectrometry (modern approach)
  • Culture of aerobic bacteria: standard incubation at 35–37°C, ambient air; shaker culture for aeration in liquid media
  • Culture of anaerobic bacteria: anaerobic jar (McIntosh-Fildes jar), anaerobic workstation/glove box, GasPak system; use of reducing agents in media (thioglycollate, cysteine); candle jar for microaerophiles (5% CO₂)
  • Culture preservation methods: short-term (refrigeration at 4°C, sub-culturing), medium-term (overlaying with mineral oil, drying on sand or soil), long-term (lyophilization/freeze-drying — most reliable for stock cultures; ultra-low temperature storage at −70°C or −196°C in liquid nitrogen)

Topic 7: Metabolism (15 teaching hours — joint largest topic)

  • Introduction to metabolism: anabolism vs. catabolism; energy currency (ATP); role of NAD⁺/NADH, NADP⁺/NADPH, FAD/FADH₂; high-energy phosphate bonds; free energy (ΔG°’)
  • Regulation of metabolism: enzyme regulation — allosteric regulation (feedback inhibition), covalent modification (phosphorylation/dephosphorylation); gene-level regulation — induction (lac operon) and catabolite repression; enzyme synthesis — constitutive vs. inducible enzymes
  • Glycolysis — Embden-Meyerhof-Parnas (EMP) pathway: substrate; steps; key enzymes (hexokinase, phosphofructokinase, pyruvate kinase); net yield (2 ATP, 2 NADH, 2 pyruvate per glucose); significance
  • Glycogenesis: synthesis of glycogen from glucose-1-phosphate via UDP-glucose; significance in energy storage
  • Glycogenolysis: breakdown of glycogen to glucose-1-phosphate; phosphorylase enzyme; significance
  • TCA cycle (Krebs cycle / Citric Acid cycle): entry of acetyl-CoA (from pyruvate decarboxylation — pyruvate dehydrogenase complex); 8 steps; key enzymes (citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase); net yield per acetyl-CoA (3 NADH, 1 FADH₂, 1 GTP, 2 CO₂); amphibolic role (biosynthetic precursors — α-ketoglutarate → glutamate; oxaloacetate → aspartate)
  • Pentose phosphate pathway (Hexose monophosphate shunt): two phases — oxidative (generates NADPH and ribulose-5-phosphate) and non-oxidative (interconverts sugars); significance — NADPH for biosynthesis and oxidative stress defense; ribose-5-phosphate for nucleotide synthesis
  • Entner-Doudoroff (ED) pathway: characteristic of Gram-negative aerobes (Pseudomonas, Zymomonas); key enzyme — 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase; net yield 1 ATP, 1 NADH, 1 NADPH per glucose; comparison with EMP
  • Phosphoketolase pathway (Heterolactic fermentation): Leuconostoc, Lactobacillus; produces lactate, ethanol, and CO₂; significance in food microbiology (sauerkraut, kimchi, silage)
  • Fermentative pathways: lactic acid fermentation (homofermentative — Streptococcus, Lactobacillus; heterofermentative — Leuconostoc); alcoholic fermentation (Saccharomyces — pyruvate → acetaldehyde → ethanol); mixed acid fermentation (E. coli — produces formic, acetic, lactic acid, ethanol, CO₂, H₂ — IMViC pattern); butanediol fermentation (Enterobacter, Klebsiella — produces 2,3-butanediol — VP positive); propionic acid fermentation (Propionibacterium — in Swiss cheese); butyric acid fermentation (Clostridium); significance in industrial microbiology
  • Electron transport system (ETS) / Oxidative phosphorylation: components — NADH dehydrogenase (Complex I), succinate dehydrogenase (Complex II), ubiquinone (CoQ), cytochrome bc₁ (Complex III), cytochrome c, cytochrome oxidase (Complex IV); proton motive force (PMF) — Mitchell’s chemiosmotic theory; ATP synthase (Complex V); P/O ratio — NADH (2.5 ATP), FADH₂ (1.5 ATP); total ATP yield from complete aerobic oxidation of glucose (30–32 ATP net); anaerobic electron transport (use of NO₃⁻, SO₄²⁻, fumarate as terminal electron acceptors — denitrification, sulfate reduction)

Topic 8: Microbial Genetics (15 teaching hours — joint largest topic)

  • Structure and function of prokaryotic DNA: double-stranded circular chromosome; supercoiling and topoisomerases (DNA gyrase — target of fluoroquinolones); nucleoid-associated proteins (HU, H-NS, Fis); GC content variation among bacteria; DNA replication — semi-conservative mechanism; bidirectional replication from oriC; enzymes (primase, DNA polymerase III — main replicative polymerase; DNA polymerase I — Okazaki fragment processing; DNA ligase); proofreading; Okazaki fragments on lagging strand
  • Genetic code: triplet code (codon); reading frame; 64 codons (61 sense + 3 stop — UAA, UAG, UGA); degeneracy (wobble hypothesis); start codon (AUG — methionine); universality; non-universal codons in mitochondria and some organisms
  • RNA types and functions: mRNA (message), tRNA (adaptor — anticodon loop, CCA-3′ end, aminoacyl-tRNA synthetase), rRNA (structural and catalytic — 16S rRNA for molecular taxonomy); transcription in prokaryotes — RNA polymerase (σ factor for promoter recognition; −10 and −35 elements; ρ-dependent and ρ-independent termination); translation — initiation (30S-mRNA-fMet-tRNA, 50S joining), elongation (peptidyl transferase — catalytic rRNA), termination (release factors)
  • Plasmids: definition (extrachromosomal, self-replicating circular DNA); types — F plasmid (conjugation), R plasmid (antibiotic resistance — R-factors), virulence plasmids, metabolic plasmids, Col plasmids (colicins); copy number (high-copy vs. low-copy plasmids); incompatibility groups; significance in clinical microbiology (spread of antibiotic resistance) and biotechnology (cloning vectors)
  • Bacterial recombination — Transformation: Griffith’s experiment (1928) — discovery of the “transforming principle” (S and R strains of Streptococcus pneumoniae); Avery, MacLeod and McCarty (1944) — identification of DNA as the transforming principle; mechanism of natural transformation (competence, DNA uptake, integration by recombination); artificial transformation (heat shock, electroporation); applications in genetic engineering
  • Bacterial recombination — Conjugation: discovery by Lederberg and Tatum (1946); F+ × F- mating — F plasmid transfer; Hfr (high frequency recombination) strains — F integrated into chromosome, chromosomal transfer; interrupted mating experiment — gene mapping; F’ factor formation; transfer of R plasmids (clinical significance — spread of multidrug resistance in hospital settings); distinction from sexual reproduction
  • Bacterial recombination — Transduction: discovery by Zinder and Lederberg (1952); bacteriophage as vector; generalized transduction (any chromosomal fragment packaged — lytic phage error) vs. specialized transduction (specific genes adjacent to integrated prophage — lysogenic phage, e.g., lambda phage carrying gal or bio genes); clinical significance (toxin gene transfer — Staphylococcal enterotoxin, cholera toxin)
  • Types and importance of mutation: definition; molecular basis — point mutations (transitions, transversions, frameshifts — insertion, deletion); spontaneous vs. induced mutations; chemical mutagens (base analogues — 5-bromouracil; alkylating agents — EMS; intercalating agents — acridine dyes); physical mutagens (UV radiation — pyrimidine dimers; ionizing radiation); Ames test for mutagenicity; suppressor mutations; reversion; selection of mutants (replica plating — Lederberg and Lederberg, 1952); adaptive/directed mutation (controversy); clinical importance of mutation (antibiotic resistance emergence, viral evolution, cancer)

Topic 9: Microscopy and Different Techniques of Handling of Microorganisms (8 teaching hours)

  • Light microscope: compound microscope — components (objective lenses — 4×, 10×, 40×, 100× oil immersion; eyepiece; condenser; iris diaphragm; stage; illuminator); magnification and resolution; numerical aperture (NA); Abbe criterion (resolution limit = 0.61λ/NA); working distance; use of immersion oil with oil-immersion lens; care and maintenance
  • Stereo (dissecting) microscope: low magnification (7–45×), three-dimensional image, working with live specimens and colonies; stage illumination
  • Darkfield microscope: special condenser (stops direct light, only scattered light reaches objective); used for unstained preparations — spirochetes (Treponema pallidum for syphilis diagnosis), Leptospira; limitations
  • Phase contrast microscope: converts phase differences (due to different refractive indices of cell components) into amplitude (brightness) differences; used for living unstained cells; components — annular diaphragm, phase plate; applications in cell biology and microbiology
  • Electron microscope: principles of electron optics (wavelength of electrons 100,000× shorter than visible light — far greater resolution ~0.1 nm); two types:
    • Transmission Electron Microscope (TEM): thin-sectioned specimens, heavy metal staining (osmium tetroxide, uranium acetate, lead citrate); internal ultrastructure; magnification up to 500,000×
    • Scanning Electron Microscope (SEM): surface topography; specimens coated with gold; three-dimensional surface images; magnification up to 100,000×
    • Negative staining for TEM (phosphotungstic acid, ammonium molybdate) — virus morphology
  • Fluorescence microscope: UV light source; fluorescent dyes (fluorochromes — FITC, TRITC, DAPI); direct fluorescent antibody test (DFA), indirect fluorescent antibody test (IFA); FISH (fluorescence in situ hybridization); flow cytometry
  • Types of staining methods: general principles (mordant, basic vs. acid dyes)
    • Simple staining: single dye (methylene blue, crystal violet); demonstrates morphology and arrangement
    • Gram staining: Crystal violet (primary stain) → Gram’s iodine (mordant) → acetone-alcohol (decolorizer) → safranin (counterstain); Gram-positive (purple) vs. Gram-negative (pink/red); mechanism — differential permeability of cell wall after decolorization; clinical significance in identifying bacterial infections; limitations
    • Acid-fast staining (Ziehl-Neelsen method): primary stain carbol fuchsin + heat → acid-alcohol decolorization → methylene blue counterstain; acid-fast positive (red) — Mycobacterium, Nocardia (partial); clinical significance in TB diagnosis
    • Spore staining (Schaeffer-Fulton method): malachite green + heat → safranin; spores green, vegetative cells red
    • Capsule staining (negative staining / India ink method): unstained background, capsule appears as clear halo around dark cell
    • Flagella staining: mordant-based staining to increase thickness (Leifson’s method, silver staining)
  • Aseptic techniques in microbiology: flaming inoculation loops; use of biological safety cabinets (BSC — Class I, II, III); sterilization before and after work; proper personal protective equipment (PPE); handling of infectious materials under Biosafety Levels (BSL 1–4); standard microbiological practices

Topic 10: Techniques in Control of Microorganisms (12 teaching hours)

  • Definitions: sterilization (complete elimination of all life including spores), disinfection (reduction of pathogenic organisms on inanimate surfaces), antisepsis (reduction of pathogenic organisms on living tissue), sanitization (reduction to safe public health levels), decontamination, bactericidal vs. bacteriostatic
  • Factors affecting efficacy of control agents: type and number of microorganisms, nature of material being treated, concentration/intensity of agent, duration of exposure, temperature, presence of organic matter (bioburden)
  • Physical methods — Temperature:
    • Moist heat: boiling (100°C, 10–30 min — kills vegetative cells, not endospores); pasteurization (HTST — 72°C for 15 sec; LTLT — 63°C for 30 min — kills pathogens, not all organisms); autoclaving (steam under pressure, 121°C at 15 psi for 15–20 min — standard sterilization, kills endospores); tyndallization (fractional sterilization — 100°C for 30 min on 3 consecutive days)
    • Dry heat: hot air oven (160–170°C for 1–2 hours — suitable for glassware, oils, powders; less effective than moist heat; no condensation)
    • Low temperature: refrigeration (0–7°C — bacteriostatic); freezing (inhibits growth, not sterilization); ultra-low freezing (−70°C — preservation)
  • Physical methods — Irradiation:
    • UV radiation (254 nm — optimal germicidal wavelength): mechanism (pyrimidine dimer formation — thymine-thymine dimers); applications (air disinfection, biosafety cabinets, surface decontamination); limitations (poor penetration, DNA repair mechanisms — photoreactivation)
    • Ionizing radiation (gamma rays from ⁶⁰Co, X-rays, electron beams): mechanism (direct DNA breakage, reactive oxygen species formation); applications (radiation sterilization of medical devices, food irradiation); high penetration; regulations on food irradiation in Nepal
  • Physical methods — Ultrasonication: high-frequency sound waves (15–400 kHz) create cavitation (formation and implosion of bubbles) — mechanical disruption of cell membranes; used for cell disruption in laboratory, not practical sterilization
  • Physical methods — Filtration: membrane filters (0.22 μm for sterilization of heat-labile liquids — vaccines, sera, antibiotics, culture media); HEPA filters (0.3 μm, 99.97% efficiency — air filtration in BSC and cleanrooms); depth filters; Swinex, Millipore filter units
  • Chemical methods — Disinfectants and antiseptics:
    • Halogens: chlorine (household bleach — sodium hypochlorite; mechanism — oxidation; water treatment, surface disinfection); iodine (tincture of iodine — skin antiseptic; iodophors — povidone-iodine — Betadine; mechanism — oxidation and iodination of proteins)
    • Phenolics: phenol (carbolic acid — Lister’s first antiseptic); chloroxylenol (Dettol); hexachlorophene; cresols; mechanism — protein denaturation and membrane damage; effectiveness vs. Mycobacterium and spores
    • Quaternary ammonium compounds (QACs — Quats): benzalkonium chloride, cetrimide; cationic surfactants; mechanism — membrane disruption; effective against Gram-positive, less vs. Gram-negative; not sporicidal
    • Alcohols: ethanol (70% — optimal), isopropanol (70%); mechanism — protein denaturation and membrane lipid dissolution; not sporicidal; hand rub, skin antiseptic
    • Aldehydes: formaldehyde (formalin — fixative, fumigation); glutaraldehyde (2% — high-level disinfectant for endoscopes — Cidex); sporicidal; mechanism — cross-linking of proteins and nucleic acids
    • Ethylene oxide (EtO): gaseous sterilant; alkylating agent; used for heat-sensitive equipment (catheters, plastic items); carcinogenic — requires post-sterilization aeration; validated sterilization cycles
    • Heavy metals: mercury (mercurochrome, thimerosal — vaccine preservative); silver (silver nitrate — neonatal eye prophylaxis; silver-impregnated catheters); copper (water pipes — oligodynamic action)
    • Hydrogen peroxide: 3% — wound antiseptic; 6–30% — high-level disinfectant; vapourized hydrogen peroxide (VHP) — room decontamination; mechanism — reactive oxygen species
  • Antibiotics and chemotherapeutic agents (introduction):
    • Definition: antibiotic (produced by microorganism), antimicrobial agent (synthetic or semi-synthetic); chemotherapeutic index (therapeutic dose / toxic dose)
    • Selective toxicity: basis of antibiotic action (exploit differences between prokaryotic and eukaryotic cells)
    • Major classes and mechanism of action:
      • Cell wall synthesis inhibitors: β-lactams (penicillins, cephalosporins, carbapenems, monobactams — inhibit PBPs/transpeptidases); glycopeptides (vancomycin — inhibits D-Ala-D-Ala precursor; effective vs. MRSA)
      • Protein synthesis inhibitors (30S): aminoglycosides (streptomycin, gentamicin — irreversible binding; bactericidal); tetracyclines (broad spectrum; bacteriostatic)
      • Protein synthesis inhibitors (50S): chloramphenicol (inhibits peptidyl transferase; bacteriostatic); macrolides (erythromycin, azithromycin — inhibit translocation); linezolid (oxazolidinone)
      • Nucleic acid synthesis inhibitors: fluoroquinolones (ciprofloxacin — inhibit DNA gyrase and topoisomerase IV; bactericidal); rifampicin (inhibits RNA polymerase — β subunit; key anti-TB drug); metronidazole (reduced to toxic radical in anaerobes — DNA strand breaks)
      • Cell membrane disruptors: polymyxins (colistin — reserved for MDR Gram-negatives)
    • Antibiotic resistance: mechanisms — enzymatic inactivation (β-lactamase, aminoglycoside-modifying enzymes), target alteration (PBP2a in MRSA), efflux pumps, reduced permeability (outer membrane porin loss); clinical significance — MRSA, ESBL-producing Enterobacteriaceae, carbapenem-resistant organisms (CRO), MDR-TB — particularly relevant in Nepal’s healthcare settings

Topic 11: Host-Parasite Interaction (10 teaching hours)

  • Normal microbial flora (microbiota) of the human body:
    • Concept of normal flora: resident vs. transient flora; commensals, mutualists, opportunistic pathogens
    • Sites and representative organisms: skin (Staphylococcus epidermidis, Propionibacterium acnes, Malassezia); oral cavity (Streptococcus mutans, Streptococcus viridans, Veillonella, Fusobacterium); respiratory tract (upper — Streptococcus pneumoniae, Haemophilus influenzae in some; lower — normally sterile); gastrointestinal tract (stomach — very few due to low pH; small intestine — limited; large intestine — massive — Bacteroides, Fusobacterium, Bifidobacterium, E. coli, Clostridium); urogenital tract (vagina — Lactobacillus acidophilus — protective function; urethra — Staphylococcus, Corynebacterium)
    • Beneficial roles of normal flora: protection against pathogens (colonization resistance); vitamin synthesis (K, B12); stimulation of immune system development; digestion of complex polysaccharides; gut microbiome and health
    • Harmful potential: opportunistic infections when host defenses are compromised; translocation across damaged mucosal barriers; dysbiosis and disease
  • Concept of host-parasite relationship:
    • Definitions: host, parasite, pathogen, virulence, pathogenicity, infection, disease, colonization, carrier
    • Types of host-parasite relationships: parasitism, commensalism, mutualism
    • Portals of entry: respiratory tract (droplet/aerosol — TB, influenza, COVID-19), gastrointestinal tract (fecal-oral — typhoid, cholera, polio), skin/mucous membranes (wound infection, sexually transmitted), parenteral (injection, arthropod vector)
    • Virulence factors of pathogens: adhesins and colonization factors (pili, surface proteins — enable attachment); invasion mechanisms (type III secretion systems, actin polymerization); toxins (exotoxins — protein, secreted — A-B type toxins: cholera toxin, diphtheria toxin, botulinum toxin; endotoxin — LPS of Gram-negative cell wall — triggers systemic inflammation); enzymes (hyaluronidase — spreading factor; coagulase; IgA protease; neuraminidase); antiphagocytic factors (capsule, M protein, protein A, leukocidins); intracellular survival (Mycobacterium, Brucella, Listeria)
    • Stages of infection: incubation period → prodromal period → acute illness → decline → convalescence
    • Epidemiology basics: source, reservoir, mode of transmission (contact, droplet, airborne, vehicle, vector); chain of infection; epidemic, endemic, pandemic — with Nepal examples (typhoid — endemic; COVID-19 — pandemic; cholera — epidemic outbreaks in Terai)
  • Concept of immunity: innate vs. adaptive immunity; physical and chemical barriers (skin, mucus, ciliary action, stomach acid, normal flora); cellular innate immunity (phagocytes — neutrophils, macrophages; NK cells); humoral innate immunity (complement system, lysozyme, interferons); adaptive immunity — overview of antibody-mediated (B cells, immunoglobulins) and cell-mediated immunity (T cells, cytotoxic T lymphocytes, helper T cells); memory cells and vaccination principle

Topic 12: Introduction to Virology (12 teaching hours)

  • General structure of viruses: obligate intracellular parasites; acellular nature; components — genome (DNA or RNA, single- or double-stranded, segmented or non-segmented, linear or circular), capsid (protein subunits — capsomers; protective; receptor binding), envelope (lipid bilayer derived from host membrane — present in enveloped viruses; absent in naked viruses), matrix proteins, spike glycoproteins (hemagglutinin, neuraminidase of influenza; gp120/gp41 of HIV)
  • Size of viruses: range 20–300 nm; comparison — poliovirus (28 nm), adenovirus (90 nm), herpesvirus (150–200 nm), poxvirus (300 nm)
  • Symmetry: icosahedral symmetry (adenovirus, poliovirus), helical symmetry (tobacco mosaic virus, influenza nucleocapsid), complex symmetry (poxvirus, bacteriophage T4)
  • Classification schemes of viruses:
    • ICTV classification: hierarchical taxonomy (realm, kingdom, phylum, class, order, family, genus, species)
    • Baltimore classification: based on genome type and mRNA production strategy — Group I (dsDNA — herpesviruses, adenoviruses); Group II (ssDNA — parvoviruses); Group III (dsRNA — reoviruses); Group IV (positive-sense ssRNA — coronaviruses, poliovirus, hepatitis A); Group V (negative-sense ssRNA — influenza, measles, rabies); Group VI (positive-sense ssRNA with reverse transcription — retroviruses including HIV); Group VII (dsDNA with reverse transcription — hepadnaviruses — hepatitis B)
  • Viral genetics: viral genome (compact, may be segmented or diploid); mutation rates (RNA viruses — higher due to RNA-dependent RNA polymerase lacking proofreading; DNA viruses — lower); reassortment (segmented RNA viruses — influenza pandemic mechanism); recombination; genetic shift vs. genetic drift in influenza; quasispecies concept
  • Virus-host interactions — replication cycle (general): attachment (specific receptor-ligand interaction — tropism); penetration (fusion or receptor-mediated endocytosis); uncoating (genome release); replication and transcription (variable by genome type); translation (use of host ribosomes); assembly; release (budding for enveloped; lysis for naked)
  • Bacteriophage (phage) replication — lytic vs. lysogenic cycle: T4 phage (lytic) — adsorption (tail fibers bind LPS/OMP of E. coli), injection of DNA, shutoff of host biosynthesis, early and late gene transcription, capsid assembly, lysozyme production, burst (50–200 phage per cell); Lambda phage (lysogenic) — integration of phage DNA as prophage into host chromosome; repressor protein (cI) maintaining lysogeny; induction by UV (SOS response) → lytic cycle; significance (phage therapy, transduction, horizontal gene transfer)
  • Detection of viruses: cytopathic effect (CPE) in cell culture — rounding, syncytia, inclusion bodies (Negri bodies in rabies); plaque assay (single-layer cell culture — plaque forming units, PFU/mL — quantitative); haemagglutination assay (influenza, measles — viral surface proteins agglutinate RBCs); haemagglutination inhibition test (HAI — antibody quantification); neutralization test; quantitative PCR (qPCR/RT-qPCR — most sensitive and specific molecular method)
  • Cultivation of viruses: embryonated eggs (influenza vaccine production — yolk sac, chorioallantoic membrane, allantoic cavity); cell culture (primary cells — not sub-cultivatable; diploid cell lines — WI-38, MRC-5; continuous/immortalized cell lines — Vero, HeLa, HEp-2); animal inoculation (suckling mice for arbovirus isolation)
  • Medically important viruses in Nepal context: Hepatitis B virus (HBV) — endemic in Nepal (3–5% HBsAg carriers); Hepatitis C virus (HCV); dengue virus (outbreaks in Terai — Aedes aegypti vector); Japanese encephalitis virus (JE — vaccination programme in Nepal); rabies virus (stray dog bites — major public health concern); HIV (increasing burden); influenza; COVID-19 (SARS-CoV-2)

Topic 13: Introduction to Parasitology (12 teaching hours)

  • Origin and definition: parasitology as the study of parasites (organisms living at the expense of another organism — the host); veterinary and human medical parasitology; evolutionary origin of host-parasite relationships
  • Classification of parasites: protozoa (unicellular eukaryotes) and helminths (multicellular worms — nematodes, trematodes, cestodes); ectoparasites (arthropods — lice, mites, ticks) vs. endoparasites
  • Blood parasites — Protozoa:
    • Plasmodium species: P. falciparum (malignant tertian — most dangerous), P. vivax (benign tertian — commonest in Nepal), P. malariae (quartan), P. ovale (rare in Nepal); life cycle — human host (liver schizogony → blood schizogony → gametocytes) and mosquito vector (Anopheles female — sporogony); clinical features (fever, chills, rigors, anaemia, splenomegaly); diagnosis (thick and thin blood smear — Giemsa stain; RDT; PCR); treatment (chloroquine for sensitive P. vivax; artemisinin-based combination therapy — ACT for P. falciparum); drug resistance; malaria distribution in Nepal (Terai districts — Jhapa, Kanchanpur, Bardiya)
    • Trypanosoma: T. brucei (African sleeping sickness — tsetse fly vector; not present in Nepal); T. cruzi (Chagas disease — reduviid bug; South America); clinical features
    • Leishmania: L. donovani — Kala-azar (visceral leishmaniasis — sandfly Phlebotomus vector); endemic in Nepal’s Terai — Siraha, Saptari, Mahottari, Dhanusha districts; clinical features (fever, splenomegaly, hepatomegaly, wasting, anaemia — “black fever”); diagnosis (splenic aspirate — amastigotes; rK39 RDT; PCR); treatment (sodium stibogluconate, miltefosine, amphotericin B); Nepal’s elimination target
  • Tissue parasites:
    • Toxoplasma gondii: definitive host (cat), intermediate hosts (humans, warm-blooded animals); clinical features (asymptomatic in immunocompetent; congenital toxoplasmosis; severe disease in immunocompromised — CNS toxoplasmosis in HIV/AIDS); diagnosis (serology, PCR)
    • Entamoeba histolytica: amoebic dysentery; liver abscess; distinction from commensal E. dispar
  • Intestinal parasites:
    • Giardia lamblia: cysts and trophozoites; giardiasis — watery diarrhoea, malabsorption; diagnosis (stool microscopy, antigen detection, PCR); treatment (metronidazole)
    • Entamoeba histolytica: life cycle (cyst ingested → trophozoite in colon → invasive dysentery or liver abscess → cyst excretion); diagnosis (stool microscopy — hematophagous trophozoites; serology; PCR)
    • Cryptosporidium parvum: acid-fast oocysts; watery diarrhoea in immunocompromised; waterborne outbreaks; no reliable treatment
  • Soil-transmitted helminths (STH) — major public health problem in Nepal’s rural and agricultural communities:
    • Ascaris lumbricoides: roundworm; largest intestinal nematode; life cycle (egg → larva penetrates gut → lungs → trachea → swallowed → adult in intestine → eggs in faeces); Löffler’s syndrome (pulmonary migration); intestinal obstruction; diagnosis (stool O&P); treatment (albendazole, mebendazole)
    • Trichuris trichiura: whipworm; large intestine; rectal prolapse in heavy infection; diagnosis (stool O&P — football-shaped eggs); treatment (albendazole)
    • Hookworm: Ancylostoma duodenale (Old World), Necator americanus; penetrate skin (ground itch); iron deficiency anaemia; diagnosis (stool O&P); treatment (albendazole)
    • Strongyloides stercoralis: unique — autoinfection and hyperinfection syndrome in immunocompromised; skin penetration; diagnosis (stool — rhabditiform larvae; Baermann technique); treatment (ivermectin)
    • Enterobius vermicularis (pinworm): perianal pruritus; diagnosis (Scotch tape test); children; treatment (albendazole)
  • Techniques of detection, enumeration, and identification:
    • Stool examination: direct wet mount (trophozoites, cysts, larvae, eggs); concentration techniques (formol-ether concentration, zinc sulfate flotation); staining (Lugol’s iodine, trichrome, iron haematoxylin, modified acid-fast for Cryptosporidium and Cyclospora); egg count (Kato-Katz — quantitative — stool egg count per gram)
    • Blood examination: thick and thin blood films (Giemsa stain) for blood parasites; Knott’s concentration for microfilariae; buffy coat examination
    • Serological tests: ELISA, IFA, indirect haemagglutination (IHA) for amoeba, Toxoplasma, Echinococcus antibodies; rK39 RDT for Leishmania
    • Molecular methods: PCR for species identification (Plasmodium species differentiation; Entamoeba histolytica vs. E. dispar)

Topic 14: Introduction to Mycology (12 teaching hours)

  • Origin and definition: mycology — the study of fungi; evolutionary origin (fungi are more closely related to animals than plants — share chitin); historical context — role of fungi in antibiotic discovery (Fleming’s penicillin, 1928)
  • Morphology of fungi: unicellular (yeasts — Saccharomyces cerevisiae, Candida albicans) vs. multicellular (moulds — Aspergillus, Penicillium, Rhizopus); dimorphic fungi (exist as yeast at 37°C, mould at 25°C — Histoplasma, Blastomyces, Coccidioides, Sporothrix, Candida — clinically important); hyphae (vegetative hyphae vs. aerial hyphae; septate vs. aseptate/coenocytic); mycelium; reproductive structures (conidiophores, sporangiophores, fruiting bodies)
  • Cell structure of fungi: cell wall (chitin + β-glucan — target of antifungal drugs — echinocandins inhibit β-glucan synthase); cell membrane (ergosterol — target of polyenes — amphotericin B — and azoles — fluconazole, itraconazole, voriconazole); true nucleus; mitochondria; 80S ribosomes; capsule in Cryptococcus neoformans
  • Medical classification of fungi (by site of infection):
    • Superficial mycoses: affect only the outermost layers (hair shaft, stratum corneum); Tinea versicolor (Malassezia furfur — skin discolouration, common in Nepal’s humid regions); Tinea nigra; black and white piedra
    • Cutaneous mycoses (dermatomycoses — dermatophytoses): affect skin, hair, nails; tinea capitis (scalp ringworm — Trichophyton tonsurans, Microsporum canis), tinea corporis (ringworm of body), tinea pedis (athlete’s foot — T. rubrum, T. interdigitale), tinea unguium/onychomycosis (nail infection); diagnosis (KOH preparation, Wood’s lamp, culture on Sabouraud’s dextrose agar)
    • Subcutaneous mycoses: implantation through trauma; Sporothrix schenckii (sporotrichosis — rose thorn disease; dimorphic; lymphocutaneous pattern); chromoblastomycosis; mycetoma (actinomycetoma vs. eumycetoma — not all are fungal)
    • Systemic/deep mycoses: primary pathogens (can infect immunocompetent): Histoplasma capsulatum (Ohio and Mississippi river valleys — not endemic in Nepal but relevant for travel history), Coccidioides immitis (southwestern USA), Blastomyces dermatitidis, Paracoccidioides brasiliensis; opportunistic pathogens (infect immunocompromised): Candida species (most common fungal nosocomial infection — candidaemia in ICU, oral thrush, vulvovaginal candidiasis); Aspergillus species (invasive pulmonary aspergillosis in neutropenic patients; Aspergillus fumigatus most common); Cryptococcus neoformans (cryptococcal meningitis — AIDS-defining illness; India ink staining of CSF — capsule; latex agglutination for cryptococcal antigen — CrAg); Pneumocystis jirovecii (PCP — Pneumocystis pneumonia — AIDS-defining illness; cannot be cultured; diagnosis by BAL and PCR; treated with co-trimoxazole); Mucorales (Rhizopus, Mucor, Lichtheimia — mucormycosis — angioinvasive, life-threatening in diabetics, immunocompromised; COVID-associated mucormycosis — black fungus — documented in South Asia including Nepal context)
  • Techniques of isolation, morphological identification, culture, and enumeration:
    • Direct microscopy: KOH preparation (10–20% KOH — dissolves keratin — visualize fungal elements in skin, nail, hair, sputum); India ink (negative staining for Cryptococcus capsule in CSF); calcofluor white (fluorescent stain — binds chitin — bright fluorescence under UV microscopy); Gomori’s methenamine silver (GMS — tissue sections — black fungal cells against green background); PAS stain
    • Culture: Sabouraud’s Dextrose Agar (SDA) — standard primary medium for dermatophytes and yeasts (low pH 5.6, high glucose suppresses bacteria); SDA + gentamicin + chloramphenicol (antibacterial); SDA + cycloheximide (inhibits environmental moulds — selective for dermatophytes); brain-heart infusion (BHI) agar/broth for dimorphic fungi; incubation temperature (25°C for moulds; 35–37°C for yeasts and dimorphic conversion); incubation duration (fungi grow slowly — 2–4 weeks for dermatophytes)
    • Colony morphology: growth rate, surface texture (powdery, granular, velvety, cottony), colour (front and reverse), size; key differential features for Aspergillus species (A. fumigatus — green, columnar heads; A. flavus — yellow-green; A. niger — black), Penicillium (blue-green), Rhizopus (grey-black, fast-growing, cotton-like)
    • Microscopic morphology: LPCB (Lactophenol cotton blue) mount — hyphae, conidia (asexual spores), conidiophores; type of conidiation (macroconidia, microconidia, chlamydoconidia, arthroconidia); germ tube test (positive for Candida albicans and C. dubliniensis — rapid identification); cornmeal agar for pseudohyphae and chlamydoconidia of Candida
    • Enumeration: colony-forming units (CFU/mL) by quantitative plate culture; Myco/F Lytic system for fungal blood cultures
    • Identification: MALDI-TOF mass spectrometry (rapid, accurate species identification from colony); molecular methods (ITS region sequencing — gold standard for species identification); β-D-glucan assay (serum — pan-fungal marker); galactomannan assay (serum, BAL — Aspergillus specific); Cryptococcal antigen (CrAg — serum or CSF)

MB 102 Practical Syllabus (50 Marks)

1. Microscopy and Staining Techniques

  • Parts and use of compound light microscope; use of oil-immersion lens; care and maintenance
  • Preparation of bacterial smears from broth and colony cultures; heat fixation
  • Simple staining: methylene blue, crystal violet; observation of morphology and arrangement
  • Gram staining: procedure, interpretation; Gram-positive (Staphylococcus aureus — clusters of cocci) and Gram-negative (E. coli — rod-shaped) controls; clinical isolate Gram staining
  • Acid-fast staining (Ziehl-Neelsen): procedure; positive control (Mycobacterium smegmatis — non-pathogenic), negative control; preparation of clinical sputum smears (simulated)
  • Spore staining (Schaeffer-Fulton): Bacillus subtilis or Bacillus cereus; observation of free spores and sporulating cells
  • Capsule staining (India ink negative stain): E. coli or Klebsiella pneumoniae capsule demonstration

2. Media Preparation and Sterilization

  • Preparation and sterilization of bacteriological culture media: nutrient broth, nutrient agar, MacConkey agar, blood agar; autoclave operation (121°C, 15 psi, 15–20 min)
  • Pouring agar plates aseptically; slant preparation
  • Hot air oven operation for dry heat sterilization of glassware (160°C, 60 min)
  • Membrane filtration: demonstration using Millipore/Swinex filter; filtration of heat-sensitive solutions
  • UV irradiation demonstration: Petri dish exposure experiment (sheltered vs. unsheltered colony comparison)

3. Culture Techniques and Isolation

  • Streak plate technique: quadrant streaking of mixed cultures for isolation of pure colonies; observation and description of colony morphology
  • Pour plate technique: serial dilution (10⁻¹ to 10⁻⁶) and pour plate for enumeration; calculation of CFU/mL
  • Spread plate technique: serial dilution and spread plate; comparison with pour plate
  • Isolation of bacteria from environmental samples: soil, water, air (settle plates)
  • Membrane filtration technique for water quality testing (simulated): coliform detection on m-Endo or MacConkey broth
  • Most Probable Number (MPN) method for coliform testing: 3-tube series; presumptive test (lactose broth); recording results; MPN table reading

4. Biochemical Tests for Bacterial Identification

  • Catalase test: H₂O₂ on colony — bubble formation (positive); distinguishing Staphylococcus (positive) from Streptococcus (negative)
  • Oxidase test: oxidase disc or tetramethyl-p-phenylenediamine reagent; distinguishing Pseudomonas (positive) from Enterobacteriaceae (negative)
  • IMViC tests: Indole test (Kovac’s reagent — red ring positive), Methyl Red test (red positive — mixed acid), Voges-Proskauer test (α-naphthol + KOH — red positive — butanediol), Citrate utilization (Simmons citrate agar — blue positive); IMViC pattern: E. coli (+ + – -) vs. Enterobacter (- – + +)
  • Urease test: Christensen’s urea agar — pink/magenta positive (Proteus, H. pylori concept); urease-negative organisms
  • Triple Sugar Iron (TSI) agar: inoculation (slant + stab); reading — H₂S (black precipitate), gas (cracks/displacement), acid/alkaline reactions (yellow/red in butt and slant); interpretation for Enterobacteriaceae
  • Sugar fermentation tests: phenol red broth with Durham tube — acid + gas vs. acid only vs. no change; glucose, lactose, sucrose, mannitol fermentation patterns
  • Coagulase test: slide and tube coagulase tests for Staphylococcus aureus identification

5. Antibiotic Sensitivity Testing

  • Kirby-Bauer disc diffusion method: Mueller-Hinton agar; inoculation by lawn technique; disc placement; incubation; measurement of inhibition zones; interpretation using CLSI breakpoints (Sensitive, Intermediate, Resistant)
  • Minimum Inhibitory Concentration (MIC) concept: broth microdilution demonstration (if available)
  • E-test strip demonstration for MIC determination

6. Mycology Practicals

  • Preparation of SDA plates and SDA+cycloheximide plates; inoculation with known fungal cultures
  • Colony morphology observation and description: Aspergillus niger, Aspergillus fumigatus, Penicillium species, Rhizopus species, Saccharomyces cerevisiae, Candida albicans
  • LPCB (Lactophenol cotton blue) mount preparation: observation of hyphae, conidia, conidiophores under microscope; drawing of fungal structures
  • KOH preparation: skin scraping (simulated), nail clipping — demonstration of fungal elements
  • Germ tube test: Candida albicans (positive) vs. Candida tropicalis (negative); 37°C serum incubation; 2–3 hour observation

7. Parasitology Practicals

  • Direct wet mount preparation of stool samples: identification of cysts and trophozoites (Entamoeba histolytica, Giardia lamblia) from permanent slides or simulated samples
  • Concentration technique (formol-ether sedimentation): demonstration; identification of eggs (Ascaris, hookworm, Trichuris) under microscope
  • Preparation and examination of thick and thin blood films (Giemsa-stained): identification of Plasmodium species from demonstration slides; ring forms, trophozoites, schizonts, gametocytes
  • India ink preparation for Cryptococcus capsule demonstration

The Four-Year TU B.Sc. Microbiology Programme Structure

  • First Year: MB 101 General Microbiology (Theory) + MB 102 (Practical) + Chemistry (Theory + Practical) + Zoology or Botany (Theory + Practical) + Scientific Communication (Theory) = 500 marks
  • Second Year: MB 201 Biochemistry and Microbial Biotechnology + MB 202 (Practical) + Chemistry + Zoology/Botany + Applied Statistics = 500 marks
  • Third Year: MB 301 Molecular Cell Biology + MB 302 (Practical) + MB 303 Pharmaceutical Microbiology and Quality Management (Elective I) + MB 304 Bioinformatics (Elective II) + Chemistry/Zoology/Botany = 500 marks
  • Fourth Year: Major subject courses (100 marks each × 2) + Practicals (50 marks each × 2) + Computational/Interdisciplinary Course (50 marks) + Project Work = 500 marks

Career Opportunities for TU B.Sc. Microbiology Graduates

  • Medical/clinical microbiology: medical laboratory technologist (MLT) in government and private hospitals — blood culture, urine culture, TB culture, antimicrobial susceptibility testing; Nepal’s expanding hospital and diagnostic laboratory network drives steady demand
  • Public health and epidemiology: District Public Health Offices (DPHOs), Epidemiology and Disease Control Division (EDCD), WHO Nepal country office, UNICEF, PATH Nepal — surveillance, outbreak investigation, disease control programmes (TB, malaria, Kala-azar, hepatitis)
  • Research institutions: Nepal Health Research Council (NHRC), BPKIHS, TUTH Department of Microbiology, KRIBS (Kathmandu Research Institute for Biological Sciences), ICIMOD — laboratory research, field epidemiology, molecular diagnostics
  • Pharmaceutical and food industries: quality control (QC) and quality assurance (QA) microbiologists in pharmaceutical manufacturing, biological product testing, food safety testing laboratories — growing with Nepal’s pharmaceutical sector expansion
  • Academic institutions: higher secondary level Biology/Science teaching (with BEd or education qualification); university-level teaching after M.Sc.; research faculty positions
  • Environmental and water quality monitoring: Department of Water Supply and Sewerage Management (DWSSM), Environmental laboratories, Nepal Bureau of Standards and Metrology (NBSM) — food and water quality testing
  • NGOs and INGOs: laboratory support roles in TB DOTS programs, HIV/AIDS programmes, malaria elimination programmes, antimicrobial resistance surveillance programmes
  • International opportunities: M.Sc. programmes in Medical Microbiology, Public Health Microbiology, Environmental Microbiology, Molecular Biology, and Biotechnology at TU (CDM), BPKIHS, or abroad (India, Germany, Japan, USA — government scholarships accessible)

Recommended Textbooks and References

Primary Textbooks (as listed in the official MB 101 syllabus)

  • Pelczar, M.J., Chan, E.C.S. and Krieg, N.R. Microbiology: Concepts and Applications. Tata McGraw-Hill (widely used across TU B.Sc. Microbiology programmes in Nepal — “Pelczar” is the standard reference)
  • Madigan, M.T., Martinko, J.M. and Parker, J. Brock Biology of Microorganisms. Prentice Hall International Inc., London (11th edition — comprehensive, internationally renowned)
  • Cappuccino, J.G. and Sherman, N. Microbiology: A Laboratory Manual. Benjamin Cummings Inc., California (4th edition — primary practical reference)
  • Collins, C.H., Patricia, M. and Lyne, J.M. Collins and Lyne’s Microbiological Methods. Butterworth-Heinemann, Oxford (7th edition)

Reference Books (as listed in the official MB 101 syllabus)

  • Atlas, R.N. Microbiology: Fundamentals and Applications. Macmillan Co.
  • Greenwood, D., Slack, R., Peutherer, J. and Barer, M. Medical Microbiology. Churchill Livingstone/ELBS (16th edition)

Additional Recommended References

  • Tortora, G.J., Funke, B.R. and Case, C.L. Microbiology: An Introduction. Pearson/Benjamin Cummings (accessible introductory text)
  • Murray, P.R., Rosenthal, K.S. and Pfaller, M.A. Medical Microbiology. Elsevier (comprehensive clinical reference)
  • Brooks, G.F., Carroll, K.C., Butel, J.S. and Morse, S.A. Jawetz, Melnick & Adelberg’s Medical Microbiology. McGraw-Hill (standard medical microbiology reference)
  • Levinson, W. Review of Medical Microbiology and Immunology. McGraw-Hill (concise review — useful for exam preparation)

Difference Between NEB Grade 12 and TU B.Sc. First Year Microbiology

Students entering TU B.Sc. First Year Microbiology from NEB Grade 12 Biology have some relevant background — the Grade 12 Biology syllabus includes microbial diseases (Typhoid, TB, HIV, Cholera, Influenza, Hepatitis, Candidiasis), applied biology (transgenic organisms, biofertilizers), and basic cell biology. However, the depth of treatment in MB 101 is categorically different. Where Grade 12 lists disease names, causative agents, and prevention, MB 101 requires a mechanistic understanding of pathogenicity (virulence factors, host-parasite interactions), detailed biochemical knowledge of metabolic pathways (glycolysis, TCA cycle, ETS — topics not covered in NEB Biology at all), formal genetics of microorganisms (transformation, conjugation, transduction, plasmids — far beyond what NEB covers), detailed microscopy and laboratory technique, and systematic coverage of all major groups of microorganisms (bacteria, viruses, fungi, parasites) at a university-level depth. The practical curriculum (MB 102) also involves hands-on laboratory skills — culture, staining, biochemical testing, antibiotic sensitivity testing, and mycological and parasitological examination — that have no direct equivalent in the NEB practical Biology curriculum.

Tips to Succeed in TU B.Sc. First Year Microbiology

  • Build a consistent study habit from Week 1 — microbiology is cumulative: Unlike subjects where units are relatively independent, microbiology topics build on each other. Cell structure (Topic 4) underpins metabolism (Topic 7) and antimicrobial action (Topic 10). Bacterial genetics (Topic 8) underpins understanding of antibiotic resistance (Topic 10) and viral genetics (Topic 12). Falling behind in early topics makes later topics disproportionately difficult to understand.
  • For metabolism (Topic 7 — 15 hours), focus on energy yields and pathway logic: The most frequently examined aspects of metabolism in TU papers are: the net ATP yield of EMP glycolysis (2 ATP, 2 NADH), TCA cycle (per acetyl-CoA: 3 NADH, 1 FADH₂, 1 GTP), and total aerobic oxidation of glucose (30–32 ATP); the unique features of ED pathway and phosphoketolase pathway; and the comparison of fermentative pathways (lactic, alcoholic, mixed acid, butanediol). Draw each pathway as a flow diagram with arrows, reactants, products, and cofactors.
  • Master the Gram stain thoroughly — it underpins clinical microbiology: The mechanism, procedure, interpretation, and clinical significance of Gram staining are foundational to all medical and clinical microbiology topics. Understand why Gram-positive bacteria retain crystal violet (thick peptidoglycan + teichoic acids) and why Gram-negatives decolorize (outer membrane dissolves in acetone-alcohol), not just the colour outcome.
  • Create a comparison table for genetic recombination methods: Transformation (DNA uptake), conjugation (cell-to-cell contact, F pilus), and transduction (phage-mediated) are all in Topic 8 and are frequently asked in comparison questions. A three-column table with rows for mechanism, discovery, DNA transferred, frequency, and clinical significance condenses what would otherwise be three separate memorization tasks into one structured reference.
  • Use Pelczar’s Microbiology as your primary theory reference: Pelczar, Chan, and Krieg’s Microbiology is the standard prescribed textbook for TU B.Sc. Microbiology and is widely available across Nepal. Its chapter structure closely follows the MB 101 topic sequence. For practical work, Cappuccino and Sherman’s Microbiology: A Laboratory Manual directly supports MB 102 procedures. Both should be your first resources.
  • Attend every practical session and record results meticulously: MB 102 practical skills — Gram staining, streak plate technique, TSI interpretation, Kirby-Bauer antibiotic susceptibility testing, KOH preparations, and blood film reading — cannot be learned from reading alone. These are exam-graded practical skills requiring physical repetition. In the practical exam, you will be assessed on technique, interpretation, and result recording. Students who attend all practicals and maintain clean, accurate lab notebooks consistently score better.
  • Nepal-specific public health content is examined: The scope and applications topic (Topic 3) and host-parasite interaction topic (Topic 11) explicitly require knowledge of Nepal’s major infectious disease burden — TB (highest in South Asia), Kala-azar (elimination target), hepatitis B (endemic — 3–5% carrier rate), malaria (Terai districts), dengue (increasing outbreaks), and antimicrobial resistance (growing challenge in Nepali hospitals). Including these specific Nepal references in answers on disease and epidemiology topics demonstrably improves examination scores.

Frequently Asked Questions (FAQs)

What is the official course code and title for TU B.Sc. First Year Microbiology?

The theory paper is MB 101 — General Microbiology, carrying 100 marks with a pass mark of 35. The practical paper is MB 102, carrying 50 marks with a pass mark of 20. Both must be passed independently. Total lecture hours for theory: 150.

How many topics are covered in MB 101 and what are their teaching hours?

MB 101 covers 14 topics across 150 teaching hours: History and Development (4 hrs), Classification and Nomenclature (15 hrs), Scope and Applications (7 hrs), Morphology of Bacteria (10 hrs), Growth and Physiology (8 hrs), Isolation, Enumeration, and Culture (10 hrs), Metabolism (15 hrs), Microbial Genetics (15 hrs), Microscopy and Handling Techniques (8 hrs), Control of Microorganisms (12 hrs), Host-Parasite Interaction (10 hrs), Introduction to Virology (12 hrs), Introduction to Parasitology (12 hrs), and Introduction to Mycology (12 hrs).

How many institutions offer B.Sc. Microbiology under TU in Nepal?

Approximately 25 colleges offer B.Sc. Microbiology under Tribhuvan University in Nepal, of which 8 are constituent colleges. Around 700 students graduate with a B.Sc. majoring in Microbiology from these colleges every year.

Which streams of students are eligible for B.Sc. Microbiology?

B.Sc. Microbiology at TU is open to students from the Biological Stream of NEB Grade 12 (or equivalent) with a minimum C grade in all subjects. Students must also pass the IoST entrance examination. Physics-group students are not eligible for the Microbiology-major programme.

What are the four applied branches of microbiology taught at TU B.Sc. level?

At TU B.Sc. level, all four applied branches of microbiology — Medical Microbiology, Environmental and Public Health Microbiology, Food Microbiology, and Agricultural Microbiology — are taught as an integrated discipline. At the M.Sc. level, students can specialize in Medical Microbiology or Environmental and Public Health Microbiology (the only two M.Sc. tracks currently available at most TU institutions).

What is the recommended primary textbook for MB 101?

The most widely used primary textbook for MB 101 in TU B.Sc. Microbiology programmes across Nepal is Pelczar, Chan, and Krieg’s Microbiology: Concepts and Applications (Tata McGraw-Hill). For practical work (MB 102), Cappuccino and Sherman’s Microbiology: A Laboratory Manual is the standard reference. Both are listed in the official MB 101 syllabus as prescribed textbooks.

Where can I find Microbiology notes and past papers for TU B.Sc. First Year?

You can find study resources on nneducation.com. The official MB 101 syllabus PDF is embedded at the top of this page. For topic-wise notes, microbenotes.com (Microbe Notes — authored by TU PhD microbiologist Dr. Sagar Aryal) is the most comprehensive online resource for TU B.Sc. Microbiology First Year content. Past papers for General Microbiology (MB 101) are available through EducateNepal (educatenepal.com) and the IoST TU official website (tuiost.edu.np).

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