BMS1026 Beste Growth and ID 2024 - Bacterial Growth and Identification PDF

Key questions How do bacteria grow? How can bacterial growth be measured? What are the methods used to grow bacteria? What are the different methods used to identify bacteria? How does bacterial growth differ from our growth? Binary fission...

Key questions How do bacteria grow? How can bacterial growth be measured? What are the methods used to grow bacteria? What are the different methods used to identify bacteria? How does bacterial growth differ from our growth? Binary fission In unicellular organisms' growth results in an increase in the number of cells or individuals Binary fission 1 cell →→ 2 cells Bacterial growth Time for one cell to become two is the generation time How many bacteria after 4 generations?  Bacterial growth is logarithmic N = 2n N = number of generations Arithmetic Log 1 20 2 21 4 22 8 23 16 24 32 25 If you start with 3 bacterial cells how many cells will there be after 3 generation? Organism Generation time Clostridium perfringens 10 min Escherichia coli 15-20 min Streptococcus pneumoniae 20-30 min Campylobacter jejuni 2.7 h Mycobacterium tuberculosis 18-24 h Treponema pallidum 30 h The bacterial growth curve Phases of bacterial growth Lag phase – slow growth Time required to start up cell functions Period which the chemical composition of the cells necessary for exponential growth is established Exponential phase – exponential growth Time when cells are growing at their maximum doubling time (µ max) in the conditions provided. Characterised by N = N02n Nutrients are in excess By consuming nutrients and releasing toxic chemicals the cells constantly modify the growth environment until it no longer supports rapid growth Stationary phase – numbers remain stable Stationary phase is characterised by a stable population consisting of some growing bacteria, some dying and some bacteria in stasis. Death phase – population decreases in size Cell death > cell growth  How to measure microbial growth 1. Directly By counting the bacterial cells  Microscopically  Flow cytometer (counts cells passing through a light detector)  Plate counts (cultivation) 2. Indirectly Estimating numbers based on an indirect measurement of growth  Optical density (turbidity)  Measuring respiration  Microscopy Propidium iodide (red = dead) Green fluorescent cyto9 = alive Microscopy – Light field Vibrio Microscopy – Dark field A microscope in which an object is illuminated only from the sides so that it appears bright against a dark Treponema background. Microscopy - Fluorescence Any microscope that uses fluorescence to generate an image Simple to complicated e.g. confocal microscope. Flow cytometry: Can be combined with dyes to determine numbers of viable bacteria  Viable counts Each colony forms from one bacterial cell which multiplies to become visible.  Indirect methods of measuring growth 1. Optical density (OD) Using a spectrophotometer the amount of light which can be transmitted through a bacterial culture can be measured. As the bacteria culture grows the turbidity increases and so does the OD 2. Measuring metabolic activity/respiration Respiration (e.g. production of CO2, reduction of dyes (e.g. tetrozoliem). Optical density A blank is used which contains media but no bacteria Measuring metabolic activity  Advantages Disadvantages Total count by microscopy Viable count by culture Optical density Measuring Respiration Flow cytometry How to grow bacteria (1) Broth = Liquid media oEnrichment of bacteria and for performing growth curves.  How to grow bacteria (2) Agar = solid media oUsually first step in bacterial identification. bacteria growing on a plate oObserve the colonial morphology which aids identification. oAssess purity of a culture *Each colony is the result of a single bacterial cell undergoing multiple divisions during the period of incubation How to grow bacteria (3) Sloppy agar = semi-solid agar oDemonstrating motility oPreserving bacteria Identification of bacteria 1. Macroscopic appearance of a colony Colour Shape Size Smell Effect on media (e.g. haemolysis, colour change) Colony morphology  Non-culturable bacteria Bacteria Disease Cultivation Treponema Syphilis Never been grown. pallidum Diagnosed by direct microscopy and serology Mycobacterium Leprosy Only on footpads of mice leprae and 9 banded armadillo Chlamydia Chlamydia Only within eukaryotic cells trachomatis  Microscopy – shape of the cell Coccobacillus Palisades (e.g. Diplobacillus E.g. chlamydia Rod corynebacteria) (e.g. bacillus) (e.g. moraxella) Cocci Diplococci (e.g. Chains pneumococcus) (e.g. streptococci) Bunch of grapes (staphylococci) Comma shaped (e.g. vibrios) Spiral-many turns (e.g. spirochaetes) Spiral-few turns (e.g. spirillium)  1. Identification by staining then microscopy Simple stain (usually one colour) Negative stain (good for capsules) Fluorescent Differential  Biochemical tests Analysis of sugar fermentation products (acids, alcohol, gas, etc) Ability to utilise specific sugars Analysis of enzymes Growth under aerobic and anaerobic conditions The API Catalase test Fermentation of sugars Identification of bacteria using molecular techniques ???? ???? Identification of bacteria using mass spectrometry Matrix Assisted Laser Desorption Time of Flight. Can be used to identify bacteria directly from clinical samples (e.g. blood).  What you need to remember Bacteria don’t get bigger they divide (or do they?). Growth rates differ enormously. Growth can be measured indirectly and directly Microscopy can differentiate bacteria. Biochemistry can be useful but.. The modern diagnostic microbiology laboratory frequently uses molecular and mass spectrometry methods to identify bacteria.