Assimilate basic laboratory and culture techniques
Media preparation and sterilization
Develop aseptic transfer skills
Perform a streak-plate
Understand the reason for negative staining
Perform thin smear and negative stain
Appreciate the simplicity of the simple stain
Complete smear preparation and simple staining
Culture media
Food for your microbes
Types of media:
Liquid (broth): propagation of large numbers
Semisolid: supports motility and anaerobic growth
Solid: used for colony appearance, pure culture isolation, and storage
Media categories by composition:
Chemically defined (synthetic): known amounts of pure chemicals; suitable for autotrophic or non-fastidious organisms
Complex (non-synthetic): unknown chemical composition; supports broad nutritional requirements; suitable for fastidious organisms
Concept of sterility
The state of being free from biological contaminants
Absolutely essential for the preparation of microbiological culture media
Your microorganism’s life depends on it!
Sterilization
Process by which all living cells, spores, and acellular entities are either destroyed or removed
Methods include autoclaving, dry-heat sterilization, filtration
Disinfection: the killing, inhibition, or removal of microorganisms that may cause disease
Sanitization: reduction of the microbial population to levels that are considered safe by public health standards
Importance of sterility
In the lab setting:
Contaminated cultures can ruin experiments
Confounds experimental results
Renders data interpretation meaningless
In the real world:
Misinterpretation of specimen samples
Delayed or incorrect diagnoses
Wound and surgical infections
Tainted pharmaceutical products
Impacts on time, money, health
Ensuring sterility
Aseptic technique: a set of procedures followed to ensure safety and prevent microbial contamination
Underpins all work in microbiology!
General rules for aseptic technique…
Make transfers over a disinfected surface
Start operations only when all apparatus and materials are within immediate reach
Complete all operations as quickly as possible, but without any hurry
Vessels must be open for the minimum amount of time possible
General rules for aseptic technique… (continued)
While vessels are open, all work must be done close to a Bunsen burner flame where air currents are drawn upwards
On opening a test tube or bottle, the neck must be immediately warmed by flaming with the vessel held as near to horizontal as possible and so that any movement of air is outwards from the vessel
All items which come into contact with microorganisms must be sterilized before and after each such exposure
Nature is a melting pot of microbes
Bacteria often grow in populations containing many different species
How can we study and characterize just one specific species from the mix?
Divide and conquer! Clinical labs require growth of pure cultures prior to performing biochemical tests and identifying a suspected microbe
Isolation techniques
Based on the notion that if a bacterial cell is separated from other cells and given ample room for growth, it will form a colony
Large number of cells growing as discrete entities on solid medium
Well-isolated colonies are the foundation for pure culture methods
Streak-plate technique
Bacterial mixture is streaked over the agar surface
Establishes a dilution gradient
Achieve isolated colonies
Assume that each colony represents outgrowth from a single bacterial cell, thus a clone of a pure culture
Streak-plate technique (practical description)
Apply, flame, heavy growth
Apply loopful of culture
Allows discrete (start) colonies to form
Apply flame again to advance the streak
Progress from heavy to light growth to achieve isolation