PHRM321: Bacterial Growth Requirements

• Institutional Pharmaceutics (PHRM321) is taught by Mbuso Faya, PhD, at the Discipline of Pharmaceutical Sciences, UKZN, in South Africa

Growth Requirements

• All organisms require a source of energy. Can be either chemical or light energy.
• Chemotrophs utilize chemical energy.
• Phototrophs utilize light energy.
• Chemotrophs can be further subdivided by their electron source.
• Lithotrophs use inorganic electron sources.
• Organotrophs use organic electron sources.
• Autrotrophs use CO2 carbon source

Requirements for Bacterial Growth

• Nutritional requirements specify that carbon is needed for carbs, amino acids, peptides, and lipids.
• Nitrogen is needed for ammonium and preformed amino acids and nucleotides.
• Sulphur is needed for sulphate, but few require inorganic S or hydrogen disulphide.
• Phosphorus is needed for free inorganic phosphate.
• Minerals are also required in bacterial growth
• Growth factors and hydrogen donors and acceptors are also required.

Environmental Requirements

• Environmental growth requirements include moisture, temperature, and pH.
• Redox potential is required for the acquisitions or loss of electron
• Osmotic pressure influences the movement of water across cell membranes, affecting cell turgor. Ionic strength impacts enzyme activity and membrane stability, while carbon dioxide levels can influence microbial metabolism and growth rates. Surface tension can affect microbial interactions, and radiation exposure can induce mutagenesis or stress responses in bacteria.

Culture Media and Culture Methods

• Bacteria have to be grown (cultured) for them to be identified
• Bacteria can be grown separately (isolated) on culture media for study through appropriate procedures
• A colony is defined as a macroscopically visible collection of millions of bacteria originating from a single bacterial cell.

Culture Media

• Culture media contains the nutrients needed to sustain a microbe.
• Culture media can vary in many ingredients allowing the media to select for or against microbes.
• Glucose or glycerol are often used as carbon sources.
• Ammonium salts or nitrates function as inorganic nitrogen sources in culture media.
• Culture is the process of growing a bacterial or other biological entity in an artificial medium.
• Lysogeny broth (LB) is a nutritionally-rich medium used for bacterial growth.
• Types are based on consistency and include solid, liquid, and semi-solid.
• Types are based on constituents/ingredients and include simple, complex, synthetic (defined) and special media.

Classification of Culture Media

• Solid media contains 2% agar and is used to assess colony morphology, pigmentation, and hemolysis.
  • Nutrient agar and Blood agar are examples of solid media.
• Liquid media contains no agar.
  • It is used for inoculum preparation and blood cultures, and for the isolation of pathogens from a mixture.
  • Nutrient broth is an example of liquid media.
• Semi-solid medium contains 0.5% agar.
  • Motility medium is an example of semi-solid media.
• Simple or basal media, which serve as fundamental growth environments for various microorganisms, are exemplified by nutrient broth (NB) and nutrient agar (NA). These media contain essential nutrients such as peptone, meat extract, and sodium chloride (NaCl), which provide amino acids, vitamins, and a balanced osmotic pressure crucial for microbial growth.
  • This is present in nutrient agar: NB + 2% agar = Nutrient agar
• Complex media are media other than basal media
  • Have added ingredients that provide special nutrients.
• Synthetic, defined media are prepared from pure chemical substances with known composition.
  • An example is peptone water: 1% peptone + 0.5% NaCl in water
• Enriched media contain substances like blood, serum, or egg and are added to the basal medium.
  • They are used to grow bacteria that are exacting in their nutritional needs.
  • Blood agar and Chocolate agar are enriched media.
• Enrichment media is liquid media that is used to isolate pathogens from a mixed culture.
  • This includes inhibitory substances to suppress the unwanted organism.
  • A few examples are Selenite F Broth for Salmonella and Shigella isolation and Alkaline Peptone Water for Vibrio cholerae.
• Selective media has inhibitory substances that is added to a solid media.
  • Mac Conkey's medium isolates gram negative bacteria
  • TCBS is for V.cholerae
  • LJ medium is for M.tuberculosis
  • Wilson and Blair medium is for S. typhi
  • Potassium tellurite medium isolates Diphtheria bacilli
• Indicator media contain an indicator which changes its color when a bacterium grows in them.
  • Examples include Blood agar, Mac Conkey's medium, and Christensen's urease medium.
• Transport media is used for transporting the samples.
  • It contains delicate organisms that may not survive the time taken for transporting the specimen without a transport media.
  • Stuart's medium - non nutrient soft agar gel containing a reducing agent
  • Buffered glycerol saline - enteric bacilli
• Anaerobic media are used to grow anaerobic orgranisms
  • Examples include Robertson’s cooked meat medium and Thioglycolate medium.

Culture Methods

• Depending on the intended purpose, culture methods will be employed
• To isolate bacteria in pure cultures
• To demonstrate their properties
• To obtain sufficient growth for the preparation of antigens and for other tests
• For bacteriophage & bacteriocin susceptibility, to determine sensitivity to antibiotics and to estimate viable counts
• For maintaining stock cultures
• Streak plate method involves streaking bacteria across an agar plate to obtain isolated colonies

Types of Culture Methods

• Lawn culture provides a uniform surface growth of the bacterium.
  • It's used for bacteriophage typing, antibiotic sensitivity testing, and preparation of bacterial antigens and vaccines.
  • Lawn cultures are prepared by flooding the surface of the plate with a liquid suspension of the bacterium.
• Stab/stroke culture is made in tubes containing agar slope/slant.
  • It provides a pure growth of bacterium for slide agglutination and other diagnostic tests.
• Stab culture prepared by puncturing a suitable medium-gelatin or glucose agar with a long, straight, charged wire.
  • It is used to demonstrate gelatin liquefaction, assess oxygen requirements, and maintain stoke cultures.
• Pour plate culture are using agar medium that is melted (15 ml) and cooled to 45°C.
  • Then, 1 ml of the inoculum is added to the molten agar.
  • Mixed well and poured to a sterile petri dish and allowed to set
  • Incubated at 37°C, colonies will be distributed throughout the depth of the medium
  • Gives an estimate of the viable bacterial count in a suspension.
• Liquid cultures are inoculated by touching with a charged loop or by adding the inoculum with pipettes or syringes.
  • Useful for Blood culture, Sterility tests, and Continuous culture methods
  • It does not provide a pure culture from mixed inocula

Identification of Microorganisms

• Microscopy can be used to study the morphological analysis of microogranisms.
  • Cells are often stained to enhance cellular features
• Rich media allows the growth of a broad range of bacteria.
• Selective media allows the growth of only a narrow range of bacteria.
• Differential media contain dyes that react with the chemical processes of certain types of bacteria, allowing their identification.
• Methods to identify miicroorgansims include Bacteriocin Typing, Bacteriophage Typing and Genetic Composition: DNA homology experiments
• Koch's postulate is used for pathogenic bacteria identification.
• DNA sequencing, Riboprinter analysis and the polymerase chain reaction( PCR) are also used

Growth, Enumeration, Metabolism, Death

• Growth curve of bacteria is defined by exponential growth, stationary, and death phases.

Measurement of Microbial Growth

• Direct counts use counting chambers, electronic counters (flow cytometry), or membrane filters
• Viable counting methods: Spread and pour plate techniques, membrane filter technique, Turbidity for Most Probable Number (MPN)
• Measurement of Cell Mass: Dry Weight Analysis, Measurement of cell components such as DNA and Turbidity.
• Counting chambers are easy, inexpensive, and quick for counting both eukaryotes and prokaryotes.
  • They cannot not distinguish living from dead cells.
• Direct counts on membranes filter cells through special membrane to provide a dark background and can be stained with fluorescent dyes.
  • Dye staining helps to distinguish living from dead cells.
• Flow Cytometry measures microbial suspension forced through small orifice with a laser light beam
  • Movement of microbe through orifice impacts electric current that flows through orifice
  • Instances of disruption of current are counted and can measure size, DNA etc.
• VBNC (Viable but not culturable)

Viable Counting : Alive or Dead

• Cell activity is always a spectrum on a scale of living or dead

Viable Counting Methods

• Includes spread and pour plate techniques.
  • A diluted sample of bacteria is spread over solid agar surface or mixed with agar and poured into petri plate.
• After incubation the numbers of organisms are determined by counting the number of colonies multiplied by dilution factor.
• Results expressed as colony forming units (CFU).
• Membrane filter technique is used in water testing by trapping bacteria from aquatic samples are trapped on membranes
• It is determined by colony counting
• Dry weight is use for measuring the mass of cell and components such as DNA
• Turbidometric measures use light scattering and is quick, efficient and non destructive