Bacteriology Lab

Question 1

Common Diagnostic Techniques:

Answer 1

1. Culture: watch cell growth (mostly grown for 5 days at 37 degrees, disc changes colour due to bacteria reproducing + producing CO₂ à alter pH)
   
   1. Sterile sites: expect no bacteria (blood/CSF)
   2. on-sterile sites
2. Serology
3. Molecular techniques (PCR)
4. Antimicrobial/AB Susceptibility Testing (discs)

Question 2

Bacterium cell wall:

Answer 2

• Gram-positive = thick peptidoglycan layer à retains dye à stains purple
• Gram-negative = thin peptidoglycan layer in periplasm à retains counter-stain à red
  
  • Outer cell membrane usually produces bacterial toxins
• Peptidoglycan made up of 2 peptides:
  
  • NAM=N-acetylglucosamine
  • NAG=N-acetylmuramic acid
• (Melioidosis – common in Asian countries caused by Gram-negative)
• Have wide range of normal flora, kept in check by innate immune system
• Generally:
  
  • Gram POSITIVE = skin + soft tissue
  • Gram NEGATIVE = abdomen + urinary tract
• Can use non-selective agar plates designed to grow anything, or special plates:
• Chocolate Agar = cooked blood - certain bacteria can’t lyse RBCs; cooking releases nutrients in blood agar à allow certain bacteria to grow e.g. Haemophilus influenzae
• MacConkey Agar = designed to grow Gram negative organisms
• Neomycin agar = growth of anaerobic microorganisms
• Usually incubate for 24 hours on plate
• Where possible, try + send cultures BEFORE giving patients ABs
• However, give antibiotics ASAP in meningitis or meningococcal septicaemia

Question 3

Staphylococci Detection = Coagulase Test: Coagulase converts fibrinogen à fibrin

Answer 3

• Can cause severe infections (skin/soft tissue, endocarditis, osteomyelitis)
• Of staphylococci àeither coagulase positive or coagulase negative:
  
  • Coagulase POSITIVE = STAPHYLOCOCCUS AUREUS (potentially MRSA)
    
    • Positive result shows coagulation/clot
  • Coagulase NEGATIVE = common skin commensals of low pathogenic potential
• Coagulase is virulence factor à helps S. aureus to cause infection
• Don’t tend to cause infection unless opportunistic circumstances (e.g. central lines)
• Any coagulase negative bacteria probably contaminants from taking blood
• Can infect prosthetic material causing line, pacemaker infections

Streptococci divided into 2 groups depending on blood agar:

1. Alpha Haemolysis – incomplete haemolysis à turns agar GREEN
2. Beta Haemolysis – complete haemolysis à clears agar
3. (non-haemolytic e.g. gut enterococci)

• A-Haemolytic Streptococci e.g. S. pneumoniae à pneumonia + meningitis
• B-Haemolytic Streptococci further divided into group A + B:
  
  • Group A e.g. S. pyogenes
    
    • Causes skin + soft tissue infections (e.g. necrotising fasciitis)
    • Also causes rheumatic + scarlet fever
  • Group B e.g. S. agalactiae
    
    • Causes sepsis in young children (neonatal septicaemia)
    • Infections in diabetics (susceptible to group B streptococcal infection)
• For all these bacteria, no penicillin resistance
• Some (e.g. group A) can be aggressive, but treatable with ABs
  
  • DON’T confuse virulence with resistance

Question 4

Diarrhoea – causes: (infectious diarrhoea doesn’t usually last 4 months)

Answer 4

1. Bacteria:
   
   1. Salmonella (including S. typhi), Shigella, Campylobacter (enteric)
   2. E coli O157, C difficile, Cholera
2. Parasites: Amoeba, Giardia, Cryptosporidium
3. Viruses

Question 5

Stool Sample Investigations:

Answer 5

• Bacteria
  
  • Culture on agar plates (different pathogens have different culture requirements)
  • Only Salmonella, Shigella + Campylobacter looked for routinely.
  • C. difficile (can’t grow) – toxin detection or PCR for toxin gene:
    
    • Higher prevalence of disease/infection à higher PPV score à more reliable; do PCR to increase prevalence
• Parasites = concentrate then use special stains
• Salmonella grown on XLD (xylose lysine deoxycholate) agar plate
  
  • Salmonella CANNOT ferment xylose à red
    
    • Most other coliforms or enteric bacteria ferment xylose à yellow
  • Salmonella also forms hydrogen sulphide which forms BLACK colonies
• Campylobacter plate grown with little oxygen:
  
  • Takes long time to grow (48 hours)
  • Can survive at 42 degrees à incubate sample at 42 degrees to kill other bacteria in stool sample à put remaining campylobacter on selective agar dish
• V. cholerae grown on TCBS agar plate à green

Infectious diarrhoea doesn’t usually persist for 4 months

Question 6

Sensitivity Testing:

Answer 6

• Zone diameter breakpoints help assess if bacteria resistant or sensitive
• MIC = minimum amount of AB needed to inhibit bacterial growth in vitro after overnight incubation
• Gradient MIC strips: varying concentrations of AB à read off conc. at edge of no bacterial growth
• Disc diffusion: uses AB discs on agar plate, incubate for 24 hours à measure how far AB spreads out (look at cleared area diameter)
• MIC isn’t that useful on its own
• Set breakpoints correlate MIC with clinical success when using the antibiotic
• If bacteria have MIC below breakpoint à good correlation with clinical success if use that antibiotic
• If bacteria have MIC ABOVE breakpoint à reported as resistant

Question 7

Serology

Answer 7

• 1^st infection: IgG peaks later than IgM
• 2^nd infection: IgG rises rapidly first + higher than IgM