General bacteriology
General bacteriology for FCPS: structure, toxins, genetics, sterilisation and antiseptics
General bacteriology for FCPS Part 1: structure, growth, exotoxins and endotoxin, gene transfer, normal flora, sterilisation and antiseptics. Each method is matched to what must be killed and what the item can tolerate, with autoclave settings and safety pitfalls.
Bacterial structure
| Structure | Notes |
|---|---|
| Gram-positive wall | Thick peptidoglycan with teichoic and lipoteichoic acids; keeps crystal violet, stains purple |
| Gram-negative wall | Thin peptidoglycan in the periplasm, outer membrane with lipopolysaccharide (endotoxin) and porins; stains pink with safranin |
| Mycobacteria | Mycolic acids make them acid-fast (Ziehl–Neelsen); stain poorly with Gram |
| No cell wall | *Mycoplasma*: sterols in the membrane; untouched by penicillins and other beta-lactams |
| Capsule | Polysaccharide (*B. anthracis*: D-glutamate); antiphagocytic; seen by the Quellung reaction or India ink; basis of conjugate vaccines; encapsulated organisms threaten asplenic patients |
| Flagella, pili | Motility (H antigen); adhesion; the sex pilus carries DNA in conjugation |
| Spores | *Bacillus* and *Clostridium*; formed when nutrients run out |
| Plasmids | Extrachromosomal DNA carrying resistance and toxin genes |
| Ribosomes | 70S (30S + 50S), the target of many antibiotics |
- Why spores resist heat: the dehydrated core is packed with calcium dipicolinate, which binds water and stabilises DNA and proteins; a thick cortex and keratin-like coat add protection. Spores survive boiling (100 °C) but not autoclaving.
- Biofilm: on implants (prosthetic joints, valves, lines), organisms such as coagulase-negative staphylococci make an exopolysaccharide slime. Inside it, bacteria grow slowly and are shielded from antibiotics and phagocytes, so infection persists despite "sensitive" antibiotics; the implant usually has to come out.
Growth and culture
- Growth curve: lag phase (adapting), log (exponential) phase, stationary phase (nutrients run out, spores form), death phase. Beta-lactams kill best in the log phase, because they block wall synthesis that only actively dividing cells are doing.
- Oxygen needs: obligate aerobes (*Pseudomonas*, *Mycobacterium*, *Nocardia*, *Bordetella*); obligate anaerobes (*Clostridium*, *Bacteroides*, *Actinomyces*; lack catalase and superoxide dismutase; foul pus, gas; resist aminoglycosides, which need oxygen to enter); facultative anaerobes (most); microaerophiles (*Campylobacter*, *H. pylori*).
| Medium | Organism |
|---|---|
| Chocolate agar | *Haemophilus*, *Neisseria* |
| Thayer–Martin | *N. gonorrhoeae* |
| TCBS | *Vibrio* |
| Löwenstein–Jensen | *M. tuberculosis* |
| MacConkey | Gram-negative rods; lactose fermenters pink |
| Tellurite, Loeffler serum | *C. diphtheriae* |
| Bordet–Gengou / charcoal blood | *Bordetella pertussis* |
| BCYE | *Legionella* |
| Selenite F, Wilson–Blair | *Salmonella* |
| Sabouraud | Fungi |
- Obligate intracellular: *Chlamydia*, *Rickettsia*, viruses. *M. leprae* and *T. pallidum* cannot be cultured in vitro (exceptions to Koch postulates).
Normal flora
- Skin: *S. epidermidis*, diphtheroids. Mouth: viridans streptococci. Colon: *Bacteroides* (most numerous), *E. coli*. Vagina: lactobacilli, which ferment glycogen to lactic acid and keep the pH around 4–4.5, suppressing pathogens; losing them allows bacterial vaginosis and candida.
- Gut flora make vitamin K2. An ICU patient on long broad-spectrum antibiotics who is not eating loses both sources, so vitamin K-dependent factors fall; factor VII has the shortest half-life, so the PT/INR rises first. Antibiotics also allow *C. difficile* overgrowth.
Toxins
| Feature | Exotoxins | Endotoxin |
|---|---|---|
| Source | Gram-positive and Gram-negative; secreted | Gram-negative outer membrane; released on lysis |
| Chemistry | Protein, often A–B structure | Lipopolysaccharide; lipid A is toxic |
| Genes | Often plasmid or bacteriophage | Chromosomal |
| Potency | Very high, specific action | Lower, non-specific |
| Heat | Mostly heat-labile (except staphylococcal enterotoxin, *E. coli* ST) | Heat-stable |
| Toxoid and vaccine | Yes: formaldehyde makes a non-toxic but immunogenic toxoid (tetanus, diphtheria) | No |
| Effect | Specific (table below) | Fever, septic shock, DIC through macrophage CD14 and TLR4, IL-1, TNF |
| Exotoxin | Mechanism | Disease |
| Diphtheria toxin | ADP-ribosylates EF-2 | Pseudomembrane, myocarditis, neuropathy |
| *Pseudomonas* exotoxin A | ADP-ribosylates EF-2 | Tissue necrosis |
| Cholera toxin, *E. coli* LT | Activates Gs, raising cAMP | Watery diarrhoea |
| *E. coli* ST | Raises cGMP | Traveller's diarrhoea |
| Pertussis toxin | Inactivates Gi, raising cAMP | Whooping cough, lymphocytosis |
| Shiga, Shiga-like | Cuts 60S ribosomal RNA | Dysentery, HUS (O157:H7) |
| Tetanospasmin | Cleaves synaptobrevin in inhibitory interneurons (GABA, glycine) | Spastic paralysis, trismus, opisthotonos |
| Botulinum toxin | Cleaves SNARE proteins at the neuromuscular junction | Descending flaccid paralysis |
| *C. perfringens* alpha toxin | Lecithinase | Gas gangrene |
| *C. difficile* toxins A, B | Glucosylate Rho GTPases | Pseudomembranous colitis |
| Anthrax toxin | Oedema factor (adenylate cyclase), lethal factor | Black eschar, mediastinitis |
| TSST-1, streptococcal pyrogenic exotoxin | Superantigens: link MHC II to the T-cell receptor outside the groove, activating huge numbers of T cells | Toxic shock, scarlet fever |
| Staphylococcal enterotoxin | Preformed, heat-stable | Food poisoning in 1–6 hours |
| Exfoliative toxin | Cleaves desmoglein-1 | Scalded skin syndrome |
Bacterial genetics
| Mechanism | Transfer by | Notes |
|---|---|---|
| Transformation | Uptake of naked DNA | Pneumococcus, *H. influenzae*, *Neisseria* (Griffith experiment) |
| Transduction | Bacteriophage | Lysogenic conversion gives toxin genes: diphtheria, cholera, botulinum, Shiga, erythrogenic toxin |
| Conjugation | Sex pilus, cell contact | Spreads resistance plasmids (ESBL, carbapenemases) between Gram-negatives |
| Transposition | Transposons | Move genes between plasmid and chromosome (vanA) |
Sterilisation, disinfection and antisepsis
- Sterilisation kills all life including spores. Disinfection kills most pathogens on objects, not necessarily spores. Antisepsis is disinfection of living tissue.
- Resistance (most to least): prions > bacterial spores > mycobacteria > non-enveloped viruses > fungi > vegetative bacteria > enveloped viruses (HIV, HBV). Prions need extended autoclaving (134 °C for 18 minutes) with sodium hydroxide, or disposal of instruments.
- Spaulding classification: critical items entering sterile tissue (surgical instruments, implants) must be sterilised; semi-critical items touching mucosa (endoscopes, laryngoscope blades) need high-level disinfection; non-critical items touching intact skin (BP cuffs, beds) need cleaning or low-level disinfection.
| Method | Conditions | Uses and points |
|---|---|---|
| Autoclave (moist heat under pressure) | 121 °C, 15 psi, 15 minutes, or 134 °C for 3 minutes | Instruments, drapes, dressings, media; most reliable; biological indicator *Geobacillus stearothermophilus* spores |
| Hot air oven | 160 °C for 2 hours (180 °C for 30 minutes) | Glassware, powders, oils, sharp instruments; indicator *Bacillus atrophaeus* |
| Ethylene oxide | Alkylating gas, low temperature | Heat-sensitive items: plastics, catheters, anaesthetic face masks, heart–lung circuits; items need hours in an aeration cabinet because residues are toxic, irritant and carcinogenic |
| Gamma irradiation | Cold sterilisation | Industrial single-use items: syringes, gloves, sutures |
| Glutaraldehyde 2% | High-level disinfection in 20 minutes; sterilant after about 10 hours | Endoscopes, cystoscopes, bronchoscopes; irritant, being replaced by peracetic acid and ortho-phthalaldehyde |
| Hydrogen peroxide plasma | Low temperature | Heat-sensitive instruments |
| Filtration | 0.22 µm membranes; HEPA filters | Sera, antibiotic and vaccine solutions; theatre air |
| Pasteurisation | 63 °C for 30 minutes or 72 °C for 15 seconds | Milk (kills *Brucella*, *M. bovis*, *Salmonella*); not sterilisation |
| Boiling | 100 °C, 10–30 minutes | Disinfection only; spores survive |
| Incineration | Very high temperature | Clinical and anatomical waste |
- Testing an autoclave: the daily Bowie–Dick test in an empty pre-vacuum autoclave checks air removal and steam penetration; an unevenly changed sheet, paler in the centre, means air was trapped. Autoclave tape only shows that a pack went through a cycle, not that it is sterile. Biological spore indicators prove the kill.
Antiseptics and disinfectants
| Agent | Properties and uses |
|---|---|
| Alcohol 70% | Fast and broad but not sporicidal; hand rub and skin prep; 70% beats 100% because water is needed to denature proteins |
| Chlorhexidine (2% in 70% alcohol for skin; 4% scrub) | Disrupts membranes; persistent residual action; preferred skin prep for surgery and central lines; keep out of the middle ear (ototoxic to the cochlea), eyes and meninges; rare anaphylaxis |
| Povidone-iodine 10% | Broad, slowly sporicidal; skin and mucosa; used for ear and eye surgery instead of chlorhexidine |
| Sodium hypochlorite (bleach) | Oxidising, sporicidal; blood spills (10,000 ppm), theatre floors and surfaces after any case including HIV or HBV, *C. difficile* rooms; inactivated by organic matter, corrosive |
| Hydrogen peroxide | Foams on contact with catalase but antibacterial action in wounds is weak and brief |
| Phenolics, chloroxylenol | Environmental disinfection |
| Quaternary ammonium (cetrimide) | Weak detergent antiseptic; inactivated by soap |
| Formaldehyde | Fumigation; 10% formalin fixes tissue |
- Fire risk: alcohol-based preps are flammable. Let them dry completely and do not let them pool under the patient or in drapes, because a diathermy spark can ignite the vapour.
- Gas embolism: hydrogen peroxide syringed under pressure into a closed sinus or deep cavity releases oxygen into opened vessels: sudden hypotension, desaturation and a "mill-wheel" murmur. Never use it under pressure or in closed spaces.
- Hand hygiene: WHO five moments (before patient contact, before an aseptic task, after body fluid exposure, after patient contact, after touching surroundings). Alcohol rub is standard, but use soap and water when hands are visibly soiled and for *C. difficile* or norovirus, since alcohol does not kill spores or some non-enveloped viruses. Surgical scrub: chlorhexidine 4% or povidone-iodine 7.5%, or an alcohol surgical rub.
Remember Sterilise what enters sterile tissue, disinfect what touches mucosa, clean what touches skin.
Exam trap Autoclave cycle is 121 °C at 15 psi for 15 minutes (not 3 minutes; 3 minutes is the 134 °C cycle).
Exam trap Endotoxin is heat-stable lipid A with no toxoid; exotoxins are proteins that can be turned into toxoids.
How FCPS asks about this
- Exotoxin versus endotoxin: exotoxins are proteins, often phage- or plasmid-coded, convertible to toxoids; endotoxin is lipid A.
- Antibiotics and growth phase: beta-lactams act best in the log phase; biofilm explains persistent prosthetic infection.
- Spore heat resistance: calcium dipicolinate in the core; killed by autoclave, not boiling.
- Heat-sensitive plastics and masks: ethylene oxide, then aeration to remove toxic residues; endoscopes: 2% glutaraldehyde for 20 minutes.
- Autoclave checks: Bowie–Dick test detects poor air removal; *Geobacillus stearothermophilus* is the biological indicator.
- Antiseptic safety: chlorhexidine out of the middle ear; let alcohol dry before diathermy; hydrogen peroxide under pressure causes gas embolism.
- Toxin mechanisms: cholera raises cAMP via Gs, diphtheria blocks EF-2, tetanus blocks inhibitory interneurons, superantigens cause toxic shock.
Test yourself on this topic
5 of the 68 questions in our bank on this topic. Choose an answer to see why every option is right or wrong.
Why can obligate anaerobes such as Bacteroides fragilis not survive in air?
Answer: Lack catalase and SOD
Oxygen metabolism generates superoxide and hydrogen peroxide. Aerobes and facultative anaerobes have superoxide dismutase and catalase (or peroxidase) to destroy them; obligate anaerobes lack these, so oxygen is toxic. Anaerobic infections (Bacteroides, Clostridium, Peptostreptococcus, Actinomyces) are typically polymicrobial, foul-smelling, gas-forming and occur near mucosal surfaces or in necrotic tissue (abscesses, aspiration pneumonia, gas gangrene). Treatment: drainage plus metronidazole or clindamycin; aminoglycosides are ineffective because their uptake needs oxygen.
Obligate anaerobes
- Lack catalase and superoxide dismutase
- Foul-smelling pus, gas, abscesses
- Treat: metronidazole, clindamycin, drainage
- Aminoglycosides useless (need O₂ for uptake)
Remember Can't Breathe Air: Clostridium, Bacteroides, Actinomyces.
Why the other options are wrong
- Cannot make ATP at all They make ATP by fermentation.
- Need high CO₂ only That describes capnophiles.
- Oxygen dissolves the capsule Not the reason.
- Lack ribosomes All bacteria have ribosomes.
During revision of a deep, narrow postoperative wound sinus, hydrogen peroxide is syringed into the tract under pressure. The patient suddenly becomes hypotensive and desaturates, and a 'mill-wheel' murmur is heard. What is the most likely cause?
Answer: Oxygen gas embolism
Hydrogen peroxide is broken down by tissue catalase into water and oxygen; 1 mL of 3% solution releases about 10 mL of oxygen gas. In a closed cavity or under pressure, the gas can enter opened vessels and cause venous or paradoxical arterial gas embolism. Signs include sudden hypotension, desaturation, a fall in end-tidal CO₂ and a mill-wheel murmur. It should not be used in closed or deep spaces or under pressure.
Hydrogen peroxide
- Catalase splits it to water + O₂
- Small volume, large gas volume
- Closed cavity: gas embolism
- Avoid deep spaces and pressure
Remember Peroxide fizzes; in a closed space that gas can embolise.
Exam trap Its foaming looks like effective cleaning, but its antibacterial action in wounds is weak and short.
Why the other options are wrong
- Anaphylaxis to the antiseptic Anaphylaxis gives urticaria and bronchospasm, not a mill-wheel murmur.
- Fat embolism from the wound Follows long-bone fractures, with petechiae and confusion.
- Methaemoglobinaemia from peroxide Peroxide does not cause methaemoglobinaemia in this setting.
- Vasovagal reaction to pain Gives bradycardia without a murmur or desaturation.
A laparoscope enters sterile body tissue during surgery. Under the Spaulding classification, what processing does it need?
Answer: Sterilisation (critical item)
Spaulding classification: critical items enter sterile tissue or the vascular system (surgical instruments, implants, laparoscopes, urinary catheters) and must be sterilised; semi-critical items contact mucous membranes or non-intact skin (flexible GI endoscopes, laryngoscope blades) and need at least high-level disinfection; non-critical items touch intact skin (stethoscopes, BP cuffs, bed rails) and need low-level disinfection or cleaning. Cleaning to remove organic material must always come first.
Spaulding classification
- Critical (sterile tissue): sterilise
- Semi-critical (mucosa): high-level disinfection
- Non-critical (intact skin): low-level
- Always clean first
Remember The deeper it goes, the cleaner it must be.
Why the other options are wrong
- Low-level disinfection For non-critical items.
- High-level disinfection Semi-critical items.
- Cleaning with water only Insufficient.
- Intermediate-level disinfection For some non-critical items.
Before a vaginal hysterectomy, the vagina and perineum must be prepared. Which agent is most suitable for the vaginal mucosa?
Answer: Aqueous povidone-iodine
Alcohol-based preparations are the most effective for intact skin, but on mucous membranes they cause pain, irritation and chemical injury, and they pool in body cavities. Mucosa is prepared with aqueous solutions, such as aqueous povidone-iodine (or low-strength aqueous chlorhexidine). Pooled alcohol under drapes is also a fire risk with diathermy. Hydrogen peroxide is not a surgical preparation agent.
Preparing mucosa
- Skin: alcohol-based preps best
- Mucosa: aqueous solutions only
- Aqueous povidone-iodine common
- Alcohol pooling: fire risk
Remember No alcohol on mucosa.
Exam trap Check for iodine allergy; aqueous chlorhexidine is the alternative.
Why the other options are wrong
- Alcoholic chlorhexidine 2% Alcohol base irritates and injures mucosa.
- Alcoholic povidone-iodine Alcohol base is unsuitable for mucosa.
- 70% isopropyl alcohol Irritant to mucosa and has no residual action.
- Hydrogen peroxide 6% Caustic at this strength and not used for preparation.
After a long course of broad-spectrum antibiotics in ICU, a patient who is not eating develops a prolonged INR and bruising. Which vitamin deficiency is most likely and why?
Answer: Vitamin K: no diet, no gut flora
Vitamin K comes from green vegetables (K1) and from colonic bacteria (K2). Poor intake plus antibiotics that kill gut flora can quickly cause deficiency, reducing gamma-carboxylation of factors II, VII, IX and X (and proteins C and S), prolonging the PT/INR. IV or oral vitamin K corrects it within hours to a day. Newborns get vitamin K at birth because their gut is sterile.
Vitamin K deficiency
- Sources: diet (K1) and gut bacteria (K2)
- Antibiotics + poor intake: deficiency
- Factors II, VII, IX, X
- INR rises first
- Newborns: vitamin K at birth
Remember No greens, no gut bugs, no clotting.
Exam trap Factor VII has the shortest half-life, so PT rises first.
Why the other options are wrong
- Vitamin C: no fresh fruit Causes bleeding gums and perifollicular haemorrhages, not a raised INR.
- Vitamin B12: gastric damage Takes years to develop.
- Vitamin D: no sunlight in ICU Bone, not clotting.
- Vitamin A: fat malabsorption Night blindness.
Where this sits in the CPSP syllabus
- Paper 1: Microbiology, General bacteriology: structure, growth, toxins, genetics, sterilisation, disinfection
- Surgery & Allied, Paper 2: Drugs in surgical practice, Antiseptics and disinfectants