Introductory Microbiology

Microbiology is the study of organisms too small to see with the naked eye. Two groups dominate this unit: the bacteria of kingdom Monera, which are true cells but have no nucleus, and the viruses, which are not cells at all. They matter out of all proportion to their size — they fix the nitrogen in a lentil field in Bara, set curd in a Kathmandu kitchen, rot the jute in a Jhapa retting pit, and cause cholera, typhoid and COVID-19. The unit ends with what happens when we deliberately re-engineer them: biotechnology.

3.1 Monera

Monera is one of the five kingdoms of Whittaker's 1969 classification and holds all prokaryotes — organisms whose cells have no nuclear envelope and no membrane-bound organelles. In the modern three-domain scheme the kingdom splits into two domains, Archaea and Bacteria, while all other life forms one domain, Eukarya.

General characters

• Unicellular and prokaryotic; size usually 0.2–2 μm wide and 1–10 μm long. Mycoplasma (0.1–0.3 μm) is the smallest known living cell.
• No nuclear membrane, nucleolus, mitochondrion, plastid, ER, Golgi body, lysosome or true vacuole.
• Genetic material: one circular, double-stranded DNA molecule (the nucleoid) with no histone proteins, often plus small circular plasmids.
• Ribosomes are 70S (a 50S and a 30S subunit).
• Cell wall of peptidoglycan (murein) — a mesh of N-acetylglucosamine and N-acetylmuramic acid chains cross-linked by short peptides.
• Reproduction is asexual by binary fission; true meiosis and gametes are absent, but DNA can be exchanged by transformation, conjugation and transduction.
• They are the most widely distributed organisms on Earth — in soil, air, water, hot springs, snow, and inside other organisms.

Shape and arrangement

Bacteria are classified into four basic shapes. Because the daughter cells often stay stuck together after fission, each shape gives a set of characteristic arrangements, and the arrangement is part of the name.

Structure of a bacterial cell

Part	Structure	Function
Capsule / slime layer	outer mucilaginous polysaccharide sheath	protection, sticking to surfaces, resists drying and phagocytosis
Cell wall	rigid peptidoglycan mesh, 10–80 nm thick	shape, protection, resists osmotic bursting
Plasma membrane	lipoprotein bilayer, no cholesterol	selective transport, respiratory enzymes
Mesosome	infolding of the plasma membrane	increases surface area for respiration; helps in DNA replication
Nucleoid	one naked circular double-stranded DNA	carries all essential genes
Plasmid	small extra circular DNA	carries resistance, fertility and toxin genes; used as a vector in gene cloning
Ribosome	70S (50S + 30S), free in cytoplasm	protein synthesis
Flagellum	flagellin fibre with basal body and hook	locomotion
Pili (fimbriae)	short hollow protein tubes	attachment; sex pili transfer DNA in conjugation
Inclusions	glycogen, PHB, volutin, gas vacuoles	food and gas storage

Gram's stain (Christian Gram, 1884) divides bacteria into two great groups, and the difference is a difference of wall chemistry.

Feature	Gram-positive	Gram-negative
Colour after staining	violet / purple	pink / red
Peptidoglycan	thick, 20–80 nm, 40–90 % of wall	thin, 2–7 nm, 5–20 % of wall
Teichoic acid	present	absent
Outer membrane, lipopolysaccharide	absent	present
Periplasmic space	absent or very narrow	well developed
Sensitivity to penicillin	high	comparatively low
Examples	Bacillus subtilis, Staphylococcus aureus, Clostridium tetani	Escherichia coli, Vibrio cholerae, Salmonella typhi, Rhizobium leguminosarum

Nutrition

Mode	Energy / carbon source	Examples
Photoautotrophic	sunlight; CO₂ fixed	cyanobacteria (Nostoc), purple sulphur bacteria (Chromatium), green sulphur bacteria (Chlorobium)
Chemoautotrophic	oxidation of inorganic substances	Nitrosomonas (NH₃ → NO₂⁻), Nitrobacter (NO₂⁻ → NO₃⁻), Thiobacillus (S → SO₄²⁻)
Saprophytic	dead organic matter	Bacillus, most soil bacteria — the chief decomposers
Parasitic	living host, causes harm	Mycobacterium tuberculosis, Xanthomonas oryzae
Symbiotic	living host, mutual benefit	Rhizobium leguminosarum in the root nodules of legumes

Reproduction

1. Binary fission (amitosis) is the normal method. The DNA replicates, the cell elongates, a transverse septum grows inwards and the cell splits into two identical daughter cells. Under ideal conditions E. coli divides every 20 minutes.
2. Endospore formation is not reproduction — it is survival. Bacillus and Clostridium lay down a thick, almost dehydrated spore inside the cell that can survive boiling, drought and disinfectants for years, then germinate into a single vegetative cell.
3. Genetic recombination substitutes for sex. There are three routes: transformation — naked DNA is taken up from the medium (Griffith, 1928); conjugation — DNA passes through a sex pilus from a donor to a recipient (Lederberg and Tatum, 1946); transduction — DNA is carried from one cell to another by a bacteriophage (Zinder and Lederberg, 1952).

The growth of a bacterial population

Because one cell becomes two at each division, a bacterial population grows geometrically. If N_0 cells grow for time t with a generation time g, the number of generations is n = t/g and

Classification of Monera

Group	Wall	Distinguishing feature	Examples
Archaebacteria	no peptidoglycan; branched lipids with ether links	live in extreme habitats; oldest known prokaryotes	Methanobacterium (methanogen, in biogas and in cattle rumen), Halobacterium (salt lakes), Sulfolobus (hot acid springs)
Eubacteria — true bacteria	peptidoglycan	the ordinary heterotrophic and chemoautotrophic bacteria	Escherichia coli, Bacillus, Rhizobium, Vibrio cholerae
Eubacteria — cyanobacteria	peptidoglycan	oxygen-releasing photosynthesis with chlorophyll a	Nostoc, Anabaena, Oscillatoria, Spirulina
Mycoplasma (PPLO)	no cell wall	smallest living cells, pleomorphic; "jokers of the plant kingdom"	Mycoplasma gallisepticum; cause of little leaf of brinjal

Cyanobacteria (blue-green algae) deserve special mention. They are prokaryotic but photosynthesise like plants, using chlorophyll a together with the blue pigment c-phycocyanin and the red c-phycoerythrin. Filamentous forms such as Nostoc and Anabaena have thick-walled colourless cells called heterocysts in which atmospheric nitrogen is fixed; this is why Anabaena azollae, living inside the water fern Azolla, is used as a biofertiliser in rice fields. When nutrient-rich water warms up, cyanobacteria multiply into a foul-smelling water bloom that deoxygenates the water and kills fish.

Economic importance of bacteria

Useful role	Organism	Product or effect
Nitrogen fixation	Rhizobium leguminosarum, Azotobacter, Anabaena	converts N₂ into ammonia; raises soil fertility
Nitrification	Nitrosomonas, Nitrobacter	ammonia → nitrite → nitrate, the form roots absorb
Dairy	Lactobacillus, Streptococcus lactis	curd, cheese, dahi and mohi
Vinegar	Acetobacter aceti	ethanol → acetic acid
Antibiotics	Streptomyces griseus, S. venezuelae, Bacillus subtilis	streptomycin, chloramphenicol, subtilin
Retting	Clostridium, Pseudomonas	frees jute, flax and hemp fibres from the stem
Biogas	Methanobacterium	methane from cow dung — over 400,000 domestic plants in Nepal
Sewage treatment	mixed saprophytes	breaks organic waste into harmless minerals
Vitamins, amino acids	Pseudomonas denitrificans, Corynebacterium glutamicum	vitamin B₁₂, glutamic acid

Harmful effect	Disease	Causal bacterium
Human	cholera	Vibrio cholerae
Human	typhoid	Salmonella typhi
Human	tuberculosis	Mycobacterium tuberculosis
Human	tetanus	Clostridium tetani
Plant	bacterial blight of rice	Xanthomonas oryzae
Plant	citrus canker	Xanthomonas citri
Plant	crown gall	Agrobacterium tumefaciens
Other	food spoilage, denitrification, milk souring	many saprophytes

3.2 Virus

The word virus is Latin for poison. In 1892 the Russian scientist D. J. Iwanowski showed that the sap of a tobacco plant with mosaic disease stayed infectious after being passed through a filter fine enough to stop all bacteria. In 1898 M. W. Beijerinck named the agent contagium vivum fluidum — a living infectious fluid. In 1935 W. M. Stanley crystallised tobacco mosaic virus and proved it was nucleoprotein, for which he received the Nobel Prize in 1946.

Living and non-living characters

Living characters	Non-living characters
Contain nucleic acid, the genetic material	Can be crystallised and stored like a chemical
Multiply, giving thousands of copies	No respiration, no growth, no excretion
Show heredity and mutation	No cytoplasm, membrane, ribosome or enzyme system of their own
Are host- and tissue-specific	Inert outside the host — cannot multiply in a culture medium
Respond to heat, formalin and radiation	Obey the law of mass action, like a chemical

Viruses are therefore said to be on the borderline between the living and the non-living: living inside the host, a chemical outside it.

Structure

Every virus has two essential parts. The capsid is the protein coat, built of identical subunits called capsomeres; it protects the nucleic acid and decides which host cell the virus can attach to. The genome is one molecule (or a few pieces) of DNA or RNA, single- or double-stranded. Some animal viruses add an outer envelope of host membrane carrying viral spikes — influenza virus, HIV and SARS-CoV-2 are enveloped. Capsid plus genome is a nucleocapsid; one complete infective particle is a virion.

Notice a pleasing piece of arithmetic in TMV: three RNA nucleotides are bound by each protein subunit, so 6400/3 \approx 2133 — very close to the 2130 capsomeres actually counted.

Basis	Types	Examples
Host	plant viruses, animal viruses, bacteriophages, mycophages	TMV; rabies virus; T4 phage
Nucleic acid	dsDNA, ssDNA, dsRNA, ssRNA	T4 phage; φX174; wound tumour virus; TMV, HIV
Shape	rod (TMV), spherical/polyhedral (poliovirus), tadpole (phage), brick (variola), bullet (rabies)	—
Envelope	naked (TMV, poliovirus) or enveloped (influenza, HIV)	—

Multiplication — the lytic cycle

Two even simpler infectious agents exist. A viroid (T. O. Diener, 1971) is a short, naked circular RNA with no protein coat — potato spindle tuber viroid is the type example. A prion (S. Prusiner) is an infectious protein with no nucleic acid at all; prions cause scrapie in sheep, BSE ("mad cow disease") and Creutzfeldt–Jakob disease in humans.

Group	Disease	Virus
Plant	tobacco mosaic	TMV
Plant	bunchy top of banana	banana bunchy top virus
Plant	yellow vein mosaic of lady's finger	bhindi yellow vein mosaic virus
Human	poliomyelitis	poliovirus
Human	rabies	rabies (lyssa) virus
Human	AIDS	HIV, a retrovirus
Human	COVID-19	SARS-CoV-2

3.3 Impacts of biotechnology in microbiology

Microorganisms are the ideal factories: they are small, they grow within hours on cheap media, their genetics is simple, and a single plasmid can carry a foreign gene into millions of cells. The key tools are:

• Restriction endonucleases — "molecular scissors" that cut DNA at a specific base sequence. EcoRI, from E. coli, leaves sticky ends.
• DNA ligase — the glue that seals the cut ends together.
• Vectors — carriers that take the gene into the host: plasmids (pBR322), the Ti plasmid of Agrobacterium tumefaciens for plants, and bacteriophage λ.
• Host cells — usually Escherichia coli or the yeast Saccharomyces cerevisiae.
• Bioreactor (fermenter) — a stirred, aerated, temperature-controlled vessel in which the engineered microbes are grown by the thousand litres.

Field	What microbial biotechnology delivers	Example
Medicine	human proteins made in bacteria	human insulin (Humulin, 1982) from E. coli; human growth hormone; interferon
Vaccines	safe subunit vaccines	hepatitis B vaccine made in yeast
Diagnostics	gene-based detection	PCR (K. Mullis, 1983) for tuberculosis and SARS-CoV-2; DNA fingerprinting
Agriculture	insect-resistant crops	cry genes from Bacillus thuringiensis in Bt cotton and Bt maize
Biofertiliser	living nitrogen fixers instead of urea	Rhizobium seed inoculum, Azotobacter, Azolla–Anabaena in paddy
Biopesticide	microbes that kill pests	Bt spore sprays; Trichoderma against soil fungi
Food	single-cell protein, fermented foods	Spirulina tablets; gundruk, kinema, curd
Energy	methane from waste	Methanobacterium in household biogas plants
Environment	bioremediation and bioleaching	Pseudomonas putida digests oil spills; Thiobacillus ferrooxidans extracts copper from low-grade ore
Industry	cheap enzymes	amylase and lipase in detergents; protease in leather tanning

In Nepal the visible impacts are mainly agricultural and energy-related. Government and NGO programmes have installed more than 400,000 household biogas plants, each replacing firewood with methane from cattle dung. The Nepal Agricultural Research Council supplies Rhizobium inoculum for lentil, chickpea and soybean; tissue-culture laboratories multiply disease-free banana, potato and orchid plantlets; and the National Gene Bank at Khumaltar stores seed of indigenous crop landraces.

Against these gains stand real concerns: the escape of genes from genetically modified crops into wild relatives, the loss of local landraces when a single engineered variety is planted everywhere, the possibility of engineered pathogens being misused, patenting of genes taken from communities without their consent (biopiracy), and the cost of the technology, which keeps it in the hands of a few companies. Nepal regulates this work through its National Biosafety Framework, based on the Cartagena Protocol on Biosafety.

Answering "impacts of biotechnology"

Marks are awarded for named examples paired with the organism used. Write "human insulin produced in Escherichia coli", not "medicines are produced". If the question says impacts, give both benefits and hazards — two or three of each — or you forfeit half the marks.

Chapter summary

• Monera contains all prokaryotes: no nuclear envelope, no membrane-bound organelles, 70S ribosomes, a single naked circular DNA, and a peptidoglycan wall (absent in Mycoplasma, non-peptidoglycan in Archaebacteria).
• Bacterial shapes are coccus, bacillus, vibrio and spiral; the prefixes diplo-, strepto-, staphylo-, tetrad and sarcina name the arrangements.
• Gram-positive walls have thick peptidoglycan and teichoic acid and stain violet; Gram-negative walls have thin peptidoglycan plus an outer lipopolysaccharide membrane and stain pink.
• Reproduction is binary fission (amitosis); endospores are for survival, not reproduction; DNA is exchanged by transformation, conjugation and transduction.
• Population growth is geometric: N = N_0 \times 2^{n} with n = t/g.
• A virus is a nucleoprotein particle — one kind of nucleic acid in a capsid of capsomeres — that is inert outside its host and multiplies only inside it, by the lytic or the lysogenic cycle.
• T4 bacteriophage: icosahedral head of dsDNA, collar, contractile sheath, hollow core, base plate, tail fibres. TMV: helical rod 300 × 18 nm, 2130 capsomeres, ssRNA.
• Microbial biotechnology uses restriction enzymes, ligase, plasmid vectors and E. coli hosts to make insulin, vaccines, Bt crops, biofertilisers, biogas and bioremediation agents — with biosafety and biopiracy as the chief concerns.

Practice questions

Group A — Multiple choice (1 mark each)

1. The ribosome of a bacterial cell is of the type [1]a80Sb70Sc60Sd55S
2. Which structure allows a bacterium to transfer DNA during conjugation? [1]aflagellumbcapsulecsex pilusdmesosome
3. Nitrogen fixation in Nostoc takes place in the [1]aakinetebheterocystchormogonedgas vacuole
4. Tobacco mosaic virus contains [1]adouble-stranded DNAbsingle-stranded DNAcdouble-stranded RNAdsingle-stranded RNA
5. An infectious agent made only of protein, with no nucleic acid, is a [1]aviroidbprioncprophagedmycoplasma
6. The enzyme used as "molecular scissors" in gene cloning is [1]aDNA ligasebDNA polymerasecrestriction endonucleasedreverse transcriptase

Answers to Group A

1. (b) — prokaryotic ribosomes are 70S (50S + 30S); 80S is eukaryotic.

2. (c) — the F or sex pilus forms the conjugation tube.

3. (b) — the thick-walled colourless heterocyst holds the nitrogenase.

4. (d) — TMV has a single-stranded RNA genome of about 6400 nucleotides.

5. (b) — a prion is a proteinaceous infectious particle.

6. (c) — restriction endonucleases cut DNA at specific recognition sequences.

Group B — Short answer (4 marks each)

1. Draw a labelled diagram of a bacterial cell and state the function of the mesosome and of the plasmid. [4]
2. Differentiate between Gram-positive and Gram-negative bacteria (any four points, with one example of each). [4]
3. A culture of Escherichia coli containing 2 \times 10^{4} cells is grown for 2 hours at 37 °C, where its generation time is 20 minutes. Calculate the final number of cells, and state how many generations occurred. [4]
4. Why is a virus said to lie on the borderline between living and non-living? Give two characters of each kind. [4]

Answers to Group B

1. Diagram must show capsule, cell wall, plasma membrane, nucleoid, plasmid, 70S ribosomes, mesosome, flagellum and pili. The mesosome is an infolding of the plasma membrane that increases the surface area for respiratory enzymes and assists DNA replication and cell-wall formation. The plasmid is extra circular DNA carrying non-essential genes such as antibiotic resistance; it replicates independently and is used as a vector in genetic engineering.

3. Number of generations n = t/g = 120/20 = 6. Then

N = N_0 \times 2^{n} = 2\times10^{4} \times 2^{6} = 2\times10^{4} \times 64 = 1.28 \times 10^{6}\ \text{cells}

So six generations occur and the population reaches 1.28 \times 10^{6} cells.

4. Living: they possess nucleic acid and so show heredity and mutation, and they multiply to give thousands of progeny inside a host. Non-living: they can be crystallised and stored like a chemical, and they have no cytoplasm, no respiration and no enzyme system of their own, so they cannot grow on a nutrient medium.

Group C — Long answer (8 marks each)

1. (a) Draw a well-labelled diagram of the T4 bacteriophage. [4](b) Describe the lytic cycle of a bacteriophage, and explain how it differs from the lysogenic cycle. [4]
2. Describe the economic importance of bacteria under the headings agriculture, industry, medicine and disease, giving the scientific name of the organism in each case. [8]

Answer outline to Group C question 1

(a) Labels required: icosahedral head containing double-stranded DNA, collar, contractile tail sheath, hollow tail core, base plate, tail spikes and six long tail fibres; total length about 200 nm.

(b) Lytic cycle — (i) adsorption: tail fibres bind specific receptors on the wall of E. coli; (ii) penetration: the sheath contracts, the core pierces the wall and the DNA is injected while the empty coat (ghost) stays outside; (iii) biosynthesis (eclipse): host DNA is hydrolysed and the host machinery is diverted to make phage DNA and capsid proteins; (iv) maturation: parts assemble into complete virions; (v) lysis: endolysin dissolves the wall and 100–200 phages are released in about 25 minutes. The host always dies.

In the lysogenic cycle the injected DNA instead integrates into the bacterial chromosome as a prophage and is replicated silently with the host DNA for many generations; the host survives, and only when induced by ultraviolet light or chemicals does the prophage excise itself and enter the lytic cycle. Bacteriophage λ is the standard example.