Growth and identification of bacterial pathogens Flashcards
Gram Stain
Gram-positives and Gram-negatives
Gram positive bacteria have cell walls with a thicker layer of peptidoglycan/murein, which retains the crystal violet/iodine complex within their cells when washed with alcohol - staining purple.
Whereas, when washed with alcohol the lipopolysaccharide capsule is removed from the gram-negative cells. They can then be counterstained with safranin and appear pink.
Microbial growth
Temperature: Affects enzyme activity and membrane fluidity
Most pathogens exhibit optimal growth around physiological pH (7.4)
Nutrient Availability: Essential for biosynthesis and energy production; nutrient scarcity limits growth.
Aerobic bacteria
Anaerobic bacteria (form spores)
Phases of the Bacterial Growth Curve
Divide by binary fission
Lag Phase: Initial period of slow population growth.
Exponential Phase: Providing there is no limiting factors, so nutrients and other chemical requirements are plentiful, the number of individuals increase exponentially over time.
Stationary Phase: The number of viable individuals remains constant as the production of new cells is roughly equal to the death of others. There is sufficient resources within the environment to maintain the population.
Death Phase: When nutrients and or oxygen run out (if the bacteria are aerobes). Or, more likely, toxic waste products of the cells’ metabolism build up, then viable cell numbers will drop. The death rate is greater than the production of new cells in the population.
Clinical microbiology
Suspected infection? What is causing it?
Immunological and antigen assays (blood sample)
Antibody Detection Assays
Antigen Detection Assays
Lateral Flow Assay-
Antibodies in a sample bind to labeled antibodies on a strip to give a visual result
Molecular biology assays (PCR assays, whole-genome sequencing) (blood, urine etc)
Growth-dependent microbiology
1.Direct microscopy on clinical samples
Very cheap, very fast
2.Grow sample on agar
Not all bacteria can be grown easily
Continuous Culture
Definition: A method of culturing bacteria in which fresh medium is continuously added, and waste is removed, maintaining cells in a steady-state exponential phase.
Difference: Unlike batch culture, where growth occurs in a closed system with limited nutrients, continuous culture supports prolonged and controlled growth.
Methods for Measuring Bacterial Growth
Direct Methods:
Plate Counts: Counting colonies on agar plates.
Microscopic Counts: Counting cells under a microscope using a hemocytometer.
Indirect Methods:
Turbidity Measurement: Using a spectrophotometer to measure the cloudiness of a culture.
Metabolic Activity: Measuring the by-products of bacterial metabolism, such as CO₂ or acid production.
Advantages and Disadvantages:
Direct Methods provide actual cell counts but can be time-consuming and labor-intensive.
Indirect Methods are quicker and suitable for continuous monitoring but provide estimates and can be affected by non-living particles.
