Microbiology: Types of Cultures

• Introduction to Microbiology II (MCB 202), Lecture Two, taught by Prof. B.O. OMAFUVBE.

Types of Cultures/Cultural Methods

• Broth Cultures/Liquid Batch Cultures: Organisms are grown in liquid medium with added nutrients, but no fresh nutrients are supplied during growth.
• Agar Slope/Slant Cultures: Solid medium (e.g., nutrient agar) in test tubes or bottles is dissolved, sterilized, and cooled in a sloped position.
• Inoculum can be spread over the medium surface or applied in a thin streak using a wire loop.
• Stab Culture: Used to observe motility, gas production, and gelatin liquefaction.
• Involves inoculating tubes or bottles with about 5 mL of solid or semi-solid medium by stabbing a loaded straight wire vertically into the center.
• Semi-solid culture: Cultures are grown in a medium containing 0.02-0.3% agar to increase the viscosity but not solidify the medium completely.
• Shake Cultures: Essential for observing colony formation in deep agar cultures, especially anaerobic or microaerophilic organisms.
• Test tubes or bottles containing 15 to 20 mL of sterilized medium is cooled to 45°C
• Inoculate medium with 0.1 mL appropriately diluted inoculum, and mix by rotating between the palms of the hand
• Solidify and incubate in an upright position

Plate Cultures

• Plate cultures are divided into streak plate, pour plate and spread plate methods
• Streak Plates: Used when isolated colonies are needed for studying colonial form or separating mixed cultures.
• Solid medium is allowed to form a thin layer in a petri-dish covered with inoculum using a streaking technique.
• Method A: Inoculum is spread evenly over a small area towards the edge of the plate.
• Sterilize the wire loop and use it to make strokes from the inoculated area.
• Sterilize again, make strokes at right angles to the previous strokes.
• Sterilize once more, make a series of strokes at right angles to the previous series, towards the pool.
• Method B: Involves streaking the plate in four quadrants, sterilizing the loop between each section.
• Pour Plate (Quantitative Technique): 1 mL of the appropriate dilution of inoculum is added to 15 mL of molten agar (45-50°C) in a test tube.
• Estimation of viable cell numbers is required; inoculum is placed in a sterile petri dish (not in molten medium) and mixed with the molten agar medium.
• The agar is allowed to solidify; the plates are inverted and incubated at the appropriate temperature.
• Spread Plate Method: Dry plates of a suitable medium are used; an appropriate dilution (0.05 mL or 0.1 mL) is placed in the center of the dry plate.
• Spread the inoculum over the medium using a sterile glass spreader.
• Replace the petri dish lid, and leave for 1-2 hours to dry before inverting, then incubate at the appropriate temperature.
• Wet plates may yield confluent growth in spots due to cells growing in the liquid on the surface.

Isolation Techniques for Microorganisms

• In laboratory studies involving microbes, pure cultures of a single organism strain come from many special techniques.
• Glassware, media, and instruments must be sterile before use.
• Aseptic techniques are used, which minimize the chance of other organisms being accidentally introduced in a microbiological procedure.

Aseptic Technique Methods

• Sterilize an inoculating needle or loop by passing it through a Bunsen flame until red hot, then allow it to cool before use to transfer organisms from one source to another.
• Flame the mouth of test tubes and conical flasks immediately after removing and before replacing the cotton plug during inoculation or transfer of organisms.
• Tubes and conical flasks are held inclined during inoculation to avoid contaminants.
• Avoid unnecessary movements and discussions during organism inoculation or transfer to avoid contaminations.
• Perform isolation procedures in areas with little to no air movement, typically using an inoculating chamber; hold culture tubes or flasks close to the Bunsen flame.

Isolation of Microbes from Air (Procedure)

• Pour sterile nutrient agar (15-20 mL), cooled to about 45°C, into a sterile petri dish to prevent heavy condensation.
• Rotate the dish clockwise and anticlockwise to allow the agar to form a uniform layer.
• Allow the agar to cool, set the plates, and invert to prevent condensation drops from the lid falling into the agar.
• After drying the plates, they can be used or incubated overnight to check purity.
• Procedure Contd: Expose the agar plate to air by removing the lid for a 20 minutes and note the exact time.
• Incubate exposed plates at 30-35°C for 24-48 hours in the incubator.
• Examine plates for microbial growth in colonies, where each colony comes from a single microorganism falling onto the agar.
• Count colonies, study them, code different types, and subculture into fresh agar plates (using the streak plate technique).

Estimating Microbial Numbers in a Sample

• A bacterial population is usually reported as a total count or viable count.
• The total count is usually done by physical methods, and often the dead or indistinguishable organisms are counted in the sample.
• Physical methods: Directly counting usually by instrumentation and measurement of turbidity.
• Viable count: The cultural method assumes that a visible colony will develop from each organism or a viable unit.
• Viable counts are expressed as colony-forming units (cfu).
• Biological methods: Plate count, roll-tube count, drop count, contact plate estimations, dip-slide count, membrane filter count, and most probable number.

Maintenance or Preservation of Cultures

• It is essential to maintain pure strains obtained in pure conditions.
• Cultures maintained in the laboratory are termed stock cultures.
• Preservation Techniques:
  • Agar Slope (Slant) Cultures: Organisms can be maintained on the surface of agar slopes (e.g., nutrient agar, malt extract agar), depending on the organism.
  • Store slopes in screw-capped bottles in darkness at room temperature or in refrigerator, but they must be subcultured every 1-2 years based on the species.
• Lactic Acid Culture: Cultures of lactic acid bacteria will not grow well on solid media incubated aerobically.
  • Maintain in a liquid medium (e.g., yeast glucose chalk litmus milk or Robertson's cooked meat medium) and subculture every 2-4 months.
• Preservation Underoil: Grow cultures on agar slopes as slant culture or in stab cultures, then cover in sterile liquid paraffin or mineral oil.
  • Cultures remain viable for several years without subculturing.
• Reduced Condition: Anaerobic bacteria are best kept in a medium providing reducing conditions (e.g., Robertson's cooked meat medium) and subcultured at intervals of up to 1 year.
• Freeze-Dried Cultures: Applicable for organisms that can survive freezing; cultures are freeze-dried (lyophilized) and stored in sealed glass ampoules under vacuum.
  • Ampoules can be stored at room temperature or refrigerated and remain viable for years; useful for culture collections and dispatch.
• Deep-Freezing: Thick suspensions of bacteria are made in sterile tap water, skim milk, or 30% glycerol in 1% peptone water, distributed into vials or screw-capped bottles between -40 °C to -70 °C and can be thawed and cultured years later.

Bacterial Growth in Laboratory Conditions

• Bacteria are typically grown in broth in tubes or flasks or agar plates in the laboratory.
• These are closed or batch systems as nutrients are not renewed, nor waste removed.
• The cell population increases predictably and then declines, a pattern of stages known as a growth curve.
• Following inoculation into a liquid growth medium, the population is counted to plot a bacterial growth curve over time. This has five distinct stages: lag phase, exponential or log phase, stationary phase, death phase, and phase of prolonged decline.
• Lag Phase: A period of little or no cell division lasting from 1 hour to several days, depending on the original culture conditions and the medium transferred into.
• The cells are active metabolically, synthesizing macromolecules required for multiplication
• Log Phase: cells begin dividing at constant rate and numbers increase by the same percentage during each time interval at this phase, where cell reproduction is active.
• Cells log phase are metabolically active and are preferred for industrial purposes involving efficient production.
• Particularly sensitive to adverse conditions like radiation, exerts their effect by interfering with some important step in the growth process during the log phase.
• Stationary Phase: the rate is slow as the number of microbial deaths balances the number of new cells formed, and the population stabilizes.
• The metabolic activities of individual surviving cells is also slow at this stage; growth stops because of exhaustion of nutrients, accumulation of waste products, or harmful changes in pH.
• The length of time cells remain in the stationary phase varies depending on the species and on environmental conditions.
• Death Phase: the total number of viable cells decreases at a constant rate (number of deaths exceeds the number of new cells formed).
• Prolonged decline: There is a very gradual decrease in viable cells, lasting for days or years.
• There is dying & releasing of nutrients where the "fitter" cells are more able to cope in the deteriorating environment.
• Continuous cultures: Continuous cultures are used in research and are often required bacterial cultures.
• These cultures are maintained at exponential growth by continuing supplying fresh culture and simultaneously removing wastes and microorganisms.
• Continuous culture can be maintained in the laboratory by dripping fresh medium, wastes and spent medium in the device continually drips fresh medium into a liquid culture.