Microbiology

What to remember

• Microbes are classified as bacteria, viruses, fungi and parasites. Each group has typical shapes, stains, diseases and control methods that are asked again and again.
• Sterilisation kills all life including spores; disinfection kills most germs on objects; antisepsis is for living tissue. Know the standard temperature, pressure and time for each method.
• Immunity can be innate or acquired, and acquired immunity is active or passive. Vaccines give active immunity; antibodies given ready-made give passive immunity.

Bacteria: shape, structure and staining

Bacteria are single-celled organisms with no true nucleus (prokaryotes). The genetic material lies free in the cytoplasm. The cell is covered by a cell wall. Some bacteria have a capsule, flagella (for movement), pili (for attachment) and spores (for survival).

Bacteria are named by shape.

Arrangement helps too. Cocci in clusters are Staphylococci. Cocci in chains are Streptococci. Cocci in pairs are diplococci (for example Neisseria gonorrhoeae and Streptococcus pneumoniae).

Gram stain divides bacteria into two groups. Gram-positive bacteria keep the violet (crystal violet) colour. Gram-negative bacteria lose it and take the pink or red counter-stain (safranin). Staphylococcus and Streptococcus are Gram-positive. E. coli, Salmonella, Vibrio and Neisseria are Gram-negative. Ziehl-Neelsen stain is used for acid-fast bacilli such as the tuberculosis and leprosy bacilli. These look red on a blue background.

Bacterial growth, culture and sterilisation

A bacterial growth curve has four phases: lag, log (exponential), stationary and decline. Bacteria multiply by binary fission. Under good conditions one cell divides into two, then four, then eight. So the number after n divisions is 2ⁿ. Worked example: one cell dividing 5 times gives 2⁵ = 32 cells.

Culture media are food for bacteria in the laboratory. Nutrient agar is a simple medium. Blood agar is an enriched medium. MacConkey agar is a differential medium for intestinal bacteria. Agar is a jelly-like solid made from seaweed.

Sterilisation methods

Autoclave works because steam under pressure reaches a temperature above 100°C. It is the most reliable method for most hospital items. Indicators such as spore strips or heat-sensitive tapes show that the cycle worked.

Disinfectants and antiseptics. Chlorine (bleaching powder) is used to disinfect water and excreta. Spirit (70% alcohol) and povidone iodine are used on skin. Formalin is used to preserve specimens and for fumigation. Phenol and lysol are used to clean floors. Potassium permanganate is used for wound washing and for disinfecting wells in outbreaks. In water supplies, residual chlorine is checked with a test kit.

Viruses, fungi and parasites

Viruses are the smallest germs. They have only one kind of nucleic acid, DNA or RNA, inside a protein coat. They live and multiply only inside living cells. Antibiotics do not act on viruses. Vaccines are the main prevention. Examples: polio, measles, rabies, hepatitis A and B, dengue, chikungunya, HIV, influenza and COVID-19.

Fungi include yeasts and moulds. They cause ringworm (tinea), candidiasis and some lung infections. Candida albicans causes thrush in the mouth. Ringworm is caused by dermatophytes, not by a worm.

Parasites include protozoa and helminths (worms). Protozoal diseases: malaria (Plasmodium), amoebiasis (Entamoeba histolytica), kala-azar (Leishmania donovani) and giardiasis. Helminths: roundworm (Ascaris), hookworm (Ancylostoma), threadworm (Enterobius) and filarial worm (Wuchereria bancrofti).

Common diseases, germs, vectors and routes

A vector is a living carrier (such as a mosquito) that transmits the agent. A carrier is a person who has the germ but no symptoms. Reservoir is the place where the germ normally lives, such as humans for polio and typhoid, or animals for rabies. Incubation period is the time from entry of the germ to the first symptom.

Immunity, antibodies and vaccines

Immunity is the body's power to resist infection.

• Innate immunity is present from birth. Skin, mucous membranes, stomach acid and white cells are examples.
• Acquired immunity develops during life. It is active when the body makes its own antibodies, either after the disease or after a vaccine. It is passive when ready-made antibodies come from outside, such as from mother to baby through the placenta and breast milk, or from an injection of immunoglobulin or antiserum.

Antibodies are proteins called immunoglobulins. IgG is the most plentiful and crosses the placenta. IgM is the first antibody made in a new infection. IgA protects mucous surfaces and is found in breast milk (colostrum). IgE is linked to allergy and worm infection. White blood cells: neutrophils fight bacteria, lymphocytes (B and T cells) give specific immunity, and monocytes become macrophages.

Vaccine types: live attenuated (BCG, measles, oral polio, rotavirus), killed or inactivated (injectable polio, rabies), toxoid (tetanus, diphtheria) and subunit or recombinant (hepatitis B). Live vaccines need strict cold chain handling. Serum used for treatment, such as anti-tetanus serum or anti-snake venom, gives passive immunity.

Collecting specimens and basic laboratory points

Collect samples before starting antibiotics where possible. Use a clean, labelled, leak-proof container. Sputum for TB should be a deep cough sample, not saliva. Blood for malaria is a thick and thin smear stained with Giemsa or Leishman stain. Stool is checked for ova and cysts. Urine culture needs a clean-catch mid-stream sample. Water is tested for coliform bacteria, which show faecal pollution.

Hospital infection and safe practice

Hospital-acquired infection (nosocomial infection) is an infection a patient gets in a health facility. Common causes are unwashed hands, unclean instruments, contaminated catheters and injections given with reused needles. Prevention is hand washing, sterile technique, single-use needles and syringes, correct waste segregation and cleaning of surfaces. Wound infection germs include Staphylococcus aureus and Pseudomonas. Immunisation of the health worker against hepatitis B protects her from needle-stick injury infection. After a needle-stick injury, wash the area with soap and water, do not squeeze or suck, and report at once for advice on post-exposure treatment. Barrier methods such as gloves, masks and gowns stop germs from passing between patients and workers.

Exam traps

• Autoclave is 121°C, 15 psi, 15 minutes. Hot air oven is 160°C for 2 hours. Do not swap them.
• Boiling does not kill all spores. Autoclave does.
• Gram-positive is violet. Gram-negative is pink or red.
• Cholera germ is comma-shaped and is called a Vibrio. Typhoid germ is a rod (bacillus).
• Anopheles carries malaria. Aedes carries dengue. Culex carries filariasis and Japanese encephalitis.
• Kala-azar is spread by a sandfly, not a mosquito.
• Ringworm is a fungal disease. Antibiotics do not cure fungal or viral infection.
• Active immunity is from vaccine or disease. Passive is ready-made antibody.
• BCG, measles and oral polio are live vaccines. Tetanus and diphtheria are toxoids.
• Pasteurisation of milk is 63°C for 30 minutes (holding) or 72°C for 15 seconds (flash).

One-liners

• 1. Bacteria have no true nucleus.
• 2. Viruses need a living cell to multiply.
• 3. Binary fission: one cell gives 2ⁿ cells after n divisions.
• 4. Agar is the solidifying agent in culture media.
• 5. Ziehl-Neelsen stain shows acid-fast bacilli.
• 6. Autoclave steam sterilisation is at 121°C for 15 minutes.
• 7. Hepatitis B is a blood-borne virus.
• 8. Female Anopheles transmits malaria.
• 9. IgG crosses the placenta.
• 10. IgA is the antibody of breast milk.
• 11. A carrier has the germ but no symptoms.
• 12. Incubation period is the gap between infection and first symptom.

Practice questions

1. Which shape name is given to comma-shaped bacteria?

   1. Coccus
   2. Spirochaete
   3. Vibrio
   4. Bacillus
   Answer

   C. Vibrio

   Comma-shaped bacteria such as Vibrio cholerae are called vibrios.

2. At what temperature and time is a hot air oven normally run for sterilisation?

   1. 100°C for 10 minutes
   2. 121°C for 15 minutes
   3. 63°C for 30 minutes
   4. 160°C for 2 hours
   Answer

   D. 160°C for 2 hours

   Dry heat needs 160°C for 2 hours; 121°C for 15 minutes is the autoclave.

3. A standard autoclave cycle uses which pressure and time at 121°C?

   1. 5 psi for 2 hours
   2. 1 psi for 60 minutes
   3. 15 psi for 15 minutes
   4. 30 psi for 5 minutes
   Answer

   C. 15 psi for 15 minutes

   The standard autoclave condition is 121°C, 15 pounds per square inch, 15 minutes.

4. Gram-positive bacteria appear which colour after Gram staining?

   1. Pink
   2. Violet
   3. Green
   4. Yellow
   Answer

   B. Violet

   Gram-positive cells retain crystal violet; Gram-negative cells take the pink counter-stain.

5. Which stain is used to show the tuberculosis bacillus in sputum?

   1. Gram stain only
   2. Giemsa stain
   3. Ziehl-Neelsen stain
   4. Leishman stain
   Answer

   C. Ziehl-Neelsen stain

   Ziehl-Neelsen is the acid-fast stain; the bacilli look red on a blue background.

6. Which mosquito spreads malaria?

   1. Culex
   2. Aedes aegypti
   3. Male Anopheles
   4. Female Anopheles
   Answer

   D. Female Anopheles

   Only the female Anopheles bites and carries Plasmodium.

7. Dengue virus is spread by which mosquito?

   1. Anopheles
   2. Culex
   3. Mansonia only
   4. Aedes aegypti
   Answer

   D. Aedes aegypti

   Aedes aegypti is the vector of dengue and chikungunya.

8. The causative agent of kala-azar is

   1. Leishmania donovani
   2. Entamoeba histolytica
   3. Wuchereria bancrofti
   4. Plasmodium vivax
   Answer

   A. Leishmania donovani

   Kala-azar is caused by the protozoan Leishmania donovani, spread by sandfly.

9. Typhoid fever is caused by

   1. Vibrio cholerae
   2. Shigella dysenteriae
   3. Salmonella typhi
   4. Clostridium tetani
   Answer

   C. Salmonella typhi

   Salmonella typhi is the typhoid bacillus.

10. Which antibody is found in breast milk and protects mucous surfaces?

    1. IgA
    2. IgE
    3. IgD
    4. IgM
    Answer

    A. IgA

    Secretory IgA is present in colostrum and milk.

11. Which vaccine is a toxoid?

    1. Measles
    2. Tetanus
    3. Oral polio
    4. BCG
    Answer

    B. Tetanus

    Tetanus vaccine is made from inactivated toxin; the others are live vaccines.

12. Ringworm is caused by a

    1. Protozoan
    2. Virus
    3. Worm
    4. Fungus
    Answer

    D. Fungus

    Ringworm (tinea) is a dermatophyte fungal infection.

13. Plague is spread to humans mainly by the bite of the

    1. Sandfly
    2. Tick
    3. Body louse only
    4. Rat flea
    Answer

    D. Rat flea

    Yersinia pestis is carried by rat fleas.

14. The solidifying agent used in bacterial culture media is

    1. Gelatin only
    2. Agar
    3. Starch
    4. Peptone
    Answer

    B. Agar

    Agar from seaweed makes a firm jelly that bacteria do not digest.

15. Milk is pasteurised by the holding method at

    1. 63°C for 30 minutes
    2. 100°C for 30 minutes
    3. 121°C for 15 minutes
    4. 160°C for 2 hours
    Answer

    A. 63°C for 30 minutes

    Holding method is 63°C for 30 minutes; flash method is 72°C for 15 seconds.

16. Scrub typhus is spread by the bite of a

    1. Sandfly
    2. Mosquito
    3. Mite (chigger)
    4. Housefly
    Answer

    C. Mite (chigger)

    Orientia tsutsugamushi is carried by larval trombiculid mites.

17. Which antibody crosses the placenta to protect the newborn?

    1. IgA
    2. IgG
    3. IgM
    4. IgE
    Answer

    B. IgG

    IgG is the only immunoglobulin that crosses the placenta.

18. Which is the first antibody to appear in a new infection?

    1. IgG
    2. IgA
    3. IgM
    4. IgE
    Answer

    C. IgM

    IgM rises first; IgG follows and lasts longer.

19. One bacterial cell divides by binary fission every 20 minutes. How many cells will there be after 100 minutes if growth is not limited?

    1. 32
    2. 64
    3. 16
    4. 100
    Answer

    A. 32

    100 minutes is 5 divisions; 2⁵ = 32.

20. A culture starts with 4 cells and each cell divides 3 times. How many cells result?

    1. 16
    2. 32
    3. 24
    4. 12
    Answer

    B. 32

    4 × 2³ = 4 × 8 = 32.

21. A baby receives antibodies through breast milk. This is an example of

    1. Natural passive immunity
    2. Natural active immunity
    3. Artificial active immunity
    4. Artificial passive immunity
    Answer

    A. Natural passive immunity

    Ready-made antibodies from the mother give natural passive immunity.

22. A child develops antibodies after measles vaccination. This is

    1. Natural active immunity
    2. Artificial passive immunity
    3. Natural passive immunity
    4. Artificial active immunity
    Answer

    D. Artificial active immunity

    A vaccine makes the child's own body produce antibodies.

23. Anti-tetanus serum given after a deep wound in an unvaccinated person gives

    1. Innate immunity
    2. Artificial active immunity
    3. Artificial passive immunity
    4. Natural active immunity
    Answer

    C. Artificial passive immunity

    Ready-made antibodies from outside give passive immunity.

24. Which method is best to sterilise surgical dressings and linen in a health centre?

    1. Dipping in spirit
    2. Autoclaving
    3. Exposure to sunlight
    4. Boiling for 5 minutes
    Answer

    B. Autoclaving

    Moist heat under pressure kills spores and penetrates fabric.

25. Which is the best way to disinfect the wells in a cholera outbreak?

    1. Chlorination or potassium permanganate
    2. Boiling the well water
    3. Adding lime juice
    4. Adding salt
    Answer

    A. Chlorination or potassium permanganate

    Bleaching powder (chlorine) or permanganate is used for well disinfection.

26. A person carries typhoid bacilli in the gall bladder but has no symptoms. This person is a

    1. Reservoir animal
    2. Fomite
    3. Vector
    4. Carrier
    Answer

    D. Carrier

    A carrier harbours germs without showing disease and can spread it.

27. Glassware and oils that moist heat cannot penetrate are best sterilised by

    1. Boiling
    2. Autoclave
    3. Hot air oven
    4. Pasteurisation
    Answer

    C. Hot air oven

    Dry heat is used for glassware, powders and oils.

28. Which test water sample shows faecal pollution?

    1. High calcium
    2. Low pH only
    3. Presence of algae
    4. Presence of coliform bacteria
    Answer

    D. Presence of coliform bacteria

    Coliforms live in the gut and indicate faecal contamination.

29. Sputum collected for TB examination should be

    1. Saliva only
    2. Collected after antibiotics
    3. Mixed with water
    4. A deep cough sample in a clean container
    Answer

    D. A deep cough sample in a clean container

    Saliva is not useful; a deep cough gives the right sample.

30. Blood films for malaria parasite are usually stained with

    1. Gram stain
    2. India ink
    3. Giemsa or Leishman stain
    4. Ziehl-Neelsen stain
    Answer

    C. Giemsa or Leishman stain

    Romanowsky-type stains show Plasmodium inside red cells.

31. Which Gram-negative comma-shaped germ is found in contaminated water?

    1. Vibrio cholerae
    2. Streptococcus pyogenes
    3. Corynebacterium diphtheriae
    4. Staphylococcus aureus
    Answer

    A. Vibrio cholerae

    Cholera is water-borne; the others are Gram-positive.

32. Statements: 1. Viruses can multiply outside living cells. 2. Antibiotics are not effective against viruses. Which is/are correct?

    1. 1 only
    2. 2 only
    3. Both 1 and 2
    4. Neither 1 nor 2
    Answer

    B. 2 only

    Viruses multiply only inside living cells; antibiotics do not act on them.

33. Statements: 1. Anopheles spreads malaria. 2. Culex spreads filariasis. Which is/are correct?

    1. 1 only
    2. 2 only
    3. Both 1 and 2
    4. Neither 1 nor 2
    Answer

    C. Both 1 and 2

    Both are correct vector facts.

34. Statements: 1. BCG is a live vaccine. 2. Tetanus vaccine is a live vaccine. Which is/are correct?

    1. 1 only
    2. 2 only
    3. Both 1 and 2
    4. Neither 1 nor 2
    Answer

    A. 1 only

    BCG is live attenuated; tetanus vaccine is a toxoid.

35. Statements: 1. Autoclave works at 121°C. 2. Boiling kills all bacterial spores. Which is/are correct?

    1. 1 only
    2. 2 only
    3. Both 1 and 2
    4. Neither 1 nor 2
    Answer

    A. 1 only

    Boiling at 100°C does not reliably kill spores.

36. Statements: 1. Hepatitis A spreads by the faecal-oral route. 2. Hepatitis B spreads through blood. Which is/are correct?

    1. 1 only
    2. 2 only
    3. Both 1 and 2
    4. Neither 1 nor 2
    Answer

    C. Both 1 and 2

    Both statements are correct.

37. Statements: 1. Active immunity follows vaccination. 2. Passive immunity needs the body to make its own antibodies. Which is/are correct?

    1. 1 only
    2. 2 only
    3. Both 1 and 2
    4. Neither 1 nor 2
    Answer

    A. 1 only

    Passive immunity uses ready-made antibodies.

38. Statements: 1. Kala-azar is spread by sandfly. 2. Japanese encephalitis is spread by Culex mosquito. Which is/are correct?

    1. 1 only
    2. 2 only
    3. Both 1 and 2
    4. Neither 1 nor 2
    Answer

    C. Both 1 and 2

    Both are correct.

39. Statements: 1. Rabies is a bacterial disease. 2. Ringworm is a viral disease. Which is/are correct?

    1. 1 only
    2. 2 only
    3. Both 1 and 2
    4. Neither 1 nor 2
    Answer

    D. Neither 1 nor 2

    Rabies is viral and ringworm is fungal.

40. Match the disease with its causative agent: 1. Whooping cough, 2. Diphtheria, 3. Tetanus. Which set is correct?

    1. 1 Clostridium tetani, 2 Bordetella pertussis, 3 Corynebacterium diphtheriae
    2. 1 Bordetella pertussis, 2 Corynebacterium diphtheriae, 3 Clostridium tetani
    3. 1 Corynebacterium diphtheriae, 2 Bordetella pertussis, 3 Clostridium tetani
    4. 1 Bordetella pertussis, 2 Clostridium tetani, 3 Corynebacterium diphtheriae
    Answer

    B. 1 Bordetella pertussis, 2 Corynebacterium diphtheriae, 3 Clostridium tetani

    Pertussis, diphtheria and tetanus match their standard organisms.

41. Match the disease with its vector: 1. Plague, 2. Dengue, 3. Kala-azar. Which set is correct?

    1. 1 Aedes, 2 rat flea, 3 sandfly
    2. 1 rat flea, 2 Aedes, 3 sandfly
    3. 1 rat flea, 2 sandfly, 3 Aedes
    4. 1 sandfly, 2 Aedes, 3 rat flea
    Answer

    B. 1 rat flea, 2 Aedes, 3 sandfly

    Plague by rat flea, dengue by Aedes, kala-azar by sandfly.

42. Match the stain with its use: 1. Gram stain, 2. Ziehl-Neelsen, 3. Giemsa. Which set is correct?

    1. 1 malaria blood films, 2 acid-fast bacilli, 3 grouping bacteria
    2. 1 grouping bacteria, 2 acid-fast bacilli, 3 malaria blood films
    3. 1 grouping bacteria, 2 malaria blood films, 3 acid-fast bacilli
    4. 1 acid-fast bacilli, 2 grouping bacteria, 3 malaria blood films
    Answer

    B. 1 grouping bacteria, 2 acid-fast bacilli, 3 malaria blood films

    Gram divides bacteria; ZN shows AFB; Giemsa stains blood parasites.

43. Statements: 1. Pasteurisation by the flash method uses 72°C for 15 seconds. 2. Pasteurisation sterilises milk completely. Which is/are correct?

    1. 1 only
    2. 2 only
    3. Both 1 and 2
    4. Neither 1 nor 2
    Answer

    A. 1 only

    Pasteurisation kills most germs but does not sterilise.

44. Statements: 1. Cocci in clusters are Staphylococci. 2. Cocci in chains are Streptococci. Which is/are correct?

    1. 1 only
    2. 2 only
    3. Both 1 and 2
    4. Neither 1 nor 2
    Answer

    C. Both 1 and 2

    Both arrangements are standard.

45. Statements: 1. IgE is linked with allergy. 2. IgA is the main antibody that crosses the placenta. Which is/are correct?

    1. 1 only
    2. 2 only
    3. Both 1 and 2
    4. Neither 1 nor 2
    Answer

    A. 1 only

    IgG, not IgA, crosses the placenta.