Describe bacterial nutrition and growth. Classify culture media with examples and describe anaerobic culture.

Bacterial Nutrition and Growth Requirements

• Nutritional requirements — a carbon source, nitrogen, inorganic ions, water, and for some organisms specific growth factors that they cannot synthesise
• Classification by carbon and energy source — autotrophs use carbon dioxide; heterotrophs require organic carbon, and all medically important bacteria are heterotrophs
• Oxygen requirement — obligate aerobes (Pseudomonas, Mycobacterium); obligate anaerobes (Clostridium, Bacteroides), which lack catalase and superoxide dismutase and are killed by oxygen; facultative anaerobes (most Enterobacteriaceae, staphylococci), which grow either way; microaerophilic (Campylobacter, Helicobacter); and capnophilic organisms requiring extra carbon dioxide (Neisseria, Haemophilus, Brucella)
• Temperature — most pathogens are mesophiles growing optimally at 37°C; psychrophiles and thermophiles are of food and environmental importance
• PH — most grow best near neutrality (7.2 to 7.6); Vibrio cholerae prefers alkaline conditions (pH 8.5), which is exploited in alkaline peptone water, and lactobacilli tolerate acid

The Bacterial Growth Curve

Lag phase — no increase in number; the cells are metabolically very active, synthesising enzymes and adapting; the cell is at its largest → log (exponential) phase — growth at a constant maximum rate; cells are uniform, most metabolically active and most susceptible to antibiotics and disinfectants; this is the phase used for Gram staining and sensitivity testing → stationary phase — nutrient exhaustion and accumulation of toxic products; division equals death; spores, exotoxins and secondary metabolites are formed here → decline (death) phase — the number of viable cells falls; involution forms appear

Culture Media — Classification

• MacConkey agar is both selective and differential — the bile salts and crystal violet select against gram-positives, while lactose with neutral red differentiates fermenters from non-fermenters. It is the single most useful plate in a routine laboratory

Methods of Anaerobic Culture

• Anaerobic jar (McIntosh-Fildes) with a gas-generating sachet producing hydrogen and carbon dioxide, and a palladium catalyst; an indicator (methylene blue or resazurin) confirms anaerobiosis
• Anaerobic chamber or glove box — the most reliable, allowing plates to be handled without oxygen exposure
• Robertson cooked meat medium — the meat particles absorb oxygen and provide reducing substances; also indicates saccharolytic (pink meat) or proteolytic (black, foul-smelling) activity
• Thioglycollate broth — contains a reducing agent and permits both aerobic and anaerobic growth in one tube
• Pre-reduced media and prompt transport matter as much as the incubation method; anaerobes die during transit, which is the commonest reason they are not isolated

Identification of Bacteria from Culture

• Colony morphology — size, shape, edge, elevation, consistency, pigment and odour. Pseudomonas produces greenish pyocyanin with a characteristic fruity smell; Proteus swarms over the plate
• Haemolysis on blood agar — alpha (partial, greenish — viridans streptococci, pneumococcus); beta (complete, clear — Streptococcus pyogenes, Staphylococcus aureus); gamma (none — enterococci)
• Gram stain of the colony, and motility by hanging drop
• Biochemical reactions — catalase (separates staphylococci, positive, from streptococci, negative); coagulase (separates S. Aureus from other staphylococci); oxidase (positive in Pseudomonas, Vibrio and Neisseria; negative in Enterobacteriaceae); urease, indole, citrate, and the triple sugar iron reaction
• Serotyping with specific antisera for Salmonella, Shigella and E. Coli; and MALDI-TOF mass spectrometry, which identifies an isolate from its protein fingerprint within minutes where available

Measurement of Bacterial Growth

Applied Aspects

• Collect the specimen before antibiotics are given — a single dose can sterilise blood cultures and leave the patient without a diagnosis for the rest of the illness
• Choose the right medium and tell the laboratory what you suspect; mycobacteria, anaerobes, fungi and fastidious organisms all need specific media and prolonged incubation that will not be set up routinely
• Use a transport medium for swabs that cannot reach the laboratory promptly, and never allow a specimen for anaerobic culture to be exposed to air
• Interpret growth in the context of the site — a positive culture from a normally sterile site is significant; growth from sputum, wound swabs or urine may be colonisation or contamination
• Automated blood culture systems have transformed practice, giving results in hours rather than days, though cost limits availability in India
• Quality control of media, reagents and incubators is what makes laboratory results trustworthy, and is invisible until it fails
• Blood culture bottles must be filled to the marked volume; volume is the single greatest determinant of yield, and an under-filled bottle commonly explains a negative result in an obviously septic patient
• Incubate for long enough — brucella, HACEK organisms, fungi and mycobacteria all require extended incubation that will not happen unless the laboratory is told what is suspected
• Growth of three or more organisms from urine indicates contamination, not polymicrobial infection, and the specimen should be repeated rather than treated
• Interpret a colony count alongside the pyuria and the symptoms; bacteriuria without symptoms is usually not treated, except in pregnancy and before urological instrumentation
• Media must be quality-controlled with known organisms before use, since a batch that fails to support growth produces false negatives that nobody detects
• Selective media reduce sensitivity as well as raising specificity, so a non-selective plate is inoculated alongside where the organism is uncertain
• Automated blood culture systems have transformed turnaround, flagging positives within hours rather than days, though their cost limits availability in much of India
• Prolonged incubation of a "negative" culture is worthwhile when the clinical picture is convincing; slow growers are lost when plates are discarded at 48 hours by protocol
• Store media correctly — blood agar deteriorates, selective agents lose potency and plates dry out; the commonest cause of a laboratory "not growing anything" is the medium, not the specimen
• Record the antibiotic history on the request form, since prior therapy explains a sterile culture from an infected site and changes how the result should be read