Topic 2: Bacterial Growth, Nutrition & Culture Media
Introduction
Understanding how bacteria grow, what nutrients they require, and how they can be cultured in the laboratory is essential for diagnosing infectious diseases. For FMGE, questions on culture media, bacterial growth curve, and biochemical identification are frequently asked. This chapter covers bacterial nutrition, growth requirements, the bacterial growth curve, and the culture media used to isolate clinically important pathogens.
Bacterial Nutrition
Bacteria require specific nutrients to survive and multiply: carbon sources, nitrogen sources, minerals (sulfur, phosphorus, potassium, magnesium), vitamins (especially B-group), and water. Based on their carbon source, bacteria are classified as:
- Autotrophs — use CO₂ as the sole carbon source (e.g., Nitrosomonas)
- Heterotrophs — require organic carbon compounds (most human pathogens fall here)
- Fastidious organisms — have complex nutritional requirements (Haemophilus influenzae, Neisseria gonorrhoeae)
Based on oxygen requirements:
- Aerobes — require oxygen as terminal electron acceptor
- Anaerobes — oxygen is toxic (Clostridium, Bacteroides)
- Facultative anaerobes — can grow with or without oxygen (E. coli, Staphylococcus)
- Microaerophilic — require reduced oxygen levels (Helicobacter pylori, Campylobacter)
- Obligate aerobes — cannot survive without oxygen (Pseudomonas aeruginosa, Mycobacterium tuberculosis)
The Bacterial Growth Curve
When bacteria are inoculated into a fresh culture medium, they exhibit a predictable pattern of growth over time:
Phases of Growth
- Lag Phase — bacteria adapt to the new environment; no increase in cell number; enzymes are synthesized
- Log (Exponential) Phase — rapid, logarithmic multiplication; bacteria are most susceptible to antibiotics; generation time varies (20 min for E. coli, 24 hours for M. tuberculosis)
- Stationary Phase — nutrients begin depleting; waste products accumulate; growth rate equals death rate; bacteria produce secondary metabolites (toxins, antibiotics)
- Decline (Death) Phase — cell death exceeds multiplication; viable count decreases
Generation time is the time required for the bacterial population to double. It is shortest during the log phase and is a key characteristic used in bacterial identification.
Culture Media
Culture media are classified based on composition, consistency, and purpose.
Classification by Consistency
- Liquid media (broth) — used for enrichment, antibiotic sensitivity testing (broth dilution)
- Solid media — contains 1.5–2% agar; used for isolation of pure colonies
- Semisolid media — used for motility testing (0.5% agar)
Classification by Composition
- Synthetic/Defined media — exact chemical composition is known; used in research
- Complex/Non-synthetic media — contain natural ingredients (peptone, beef extract); used routinely in diagnostic labs
Classification by Purpose
| Media | Purpose | Example Pathogen |
|---|---|---|
| Nutrient agar | General cultivation | Most non-fastidious bacteria |
| Blood agar | Growth of fastidious organisms; hemolysis detection | Streptococcus, Haemophilus |
| Chocolate agar | Growth of fastidious organisms (heated blood) | Neisseria, Haemophilus influenzae |
| MacConkey agar | Gram-negative enteric bacteria; lactose fermentation | E. coli (pink), Salmonella (colorless) |
| SS agar | Salmonella and Shigella isolation | Salmonella typhi, Shigella |
| Wilson-Blair agar | Salmonella typhi (black colonies) | S. typhi |
| Lowenstein-Jensen (LJ) medium | Mycobacteria cultivation | M. tuberculosis |
| Robertson’s cooked meat broth | Anaerobic cultivation | Clostridium species |
| Sabouraud dextrose agar | Fungal isolation | Candida, Aspergillus |
| Thiosulfate-citrate-bile salts-sucrose (TCBS) agar | Vibrio isolation | Vibrio cholerae (yellow colonies) |
| XLD agar | Enteric pathogens | Salmonella, Shigella |
Selective and Differential Media
Selective media contain inhibitors (bile salts, antibiotics, dyes) that allow growth of only target organisms while suppressing others:
- MacConkey agar (bile salts suppress gram-positive)
- SS agar (bile salts, brilliant green suppress gram-positive and most gram-negative)
- TCBS (thiosulfate, citrate, bile — selects Vibrio)
Differential media allow identification based on biochemical properties:
- MacConkey agar differentiates lactose fermenters (pink) from non-fermenters (colorless)
- Blood agar differentiates alpha, beta, and gamma hemolysis
Bacterial Metabolism and FMGE-Relevant Biochemical Tests
Catalase Test
Distinguishes Staphylococcus (catalase-positive) from Streptococcus (catalase-negative). Bubbles form when hydrogen peroxide is added to catalase-positive bacteria.
Oxidase Test
Identifies Pseudomonas aeruginosa, Neisseria, and Vibrio (all oxidase-positive). E. coli is oxidase-negative. Uses tetramethyl-p-phenylenediamine dihydrochloride — turns purple within seconds.
Coagulase Test
Distinguishes Staphylococcus aureus (coagulase-positive) from coagulase-negative staphylococci (CoNS). Both slide and tube coagulase tests are used; tube test is more reliable.
Nitrate Reduction Test
Many Enterobacteriaceae reduce nitrate to nitrite. Pseudomonas aeruginosa is a notable non-nitrite producer (further reduces to nitrogen gas).
Indole Test
Tests for tryptophanase production. E. coli is indole-positive (red ring with Kovac’s reagent); Shigella is indole-negative.
Methyl Red (MR) and Voges-Proskauer (VP) Tests
- MR positive — mixed acid fermentation (E. coli, Salmonella)
- VP positive — butanediol fermentation (Klebsiella, Enterobacter)
Citrate Utilization Test
Klebsiella, Enterobacter, Citrobacter (KEC group) are citrate-positive; E. coli is citrate-negative.
FMGE High-Yield Points
- Chocolate agar — think Neisseria gonorrhoeae, Haemophilus influenzae (both fastidious)
- MacConkey agar — lactose fermenter = pink (E. coli, Klebsiella); non-fermenter = colorless (Salmonella, Shigella, Pseudomonas)
- TCBS (thiosulfate-citrate-bile salts-sucrose) — bright yellow colonies = Vibrio cholerae
- Lowenstein-Jensen — M. tuberculosis (rough, buff-colored colonies; 4–6 weeks growth)
- Robertson’s cooked meat broth — anaerobes, especially Clostridium
- Generation time of E. coli ≈ 20 minutes; of M. tuberculosis ≈ 24 hours — a key distinguishing feature
- Catalase test: Staph (pos) vs. Strep (neg)
- Coagulase test: S. aureus (pos) vs. CoNS (neg)
⚡ Exam tip: When choosing a culture medium, first ask: (1) What is the suspected organism? (2) Is the medium selective, differential, or both? Most FMGE questions on culture media test this logic.