Cultivation and Isolation of Bacteria NAS: You’re never too old to streak! 1Lecturer: Mr M. Zivuku

Definitions Culture A culture is the microorganisms that grow in a culture medium. To culture means to grow microorganisms in a culture medium Culture medium [pl. media] Culture media are solutions containing all of the nutrients and necessary physical growth parameters necessary for microbial growth. Note that not all microorganisms can grow in any given culture medium and, in fact, many can't grow in any known culture medium. 2Lecturer: Mr M. Zivuku

Definitions In addition to chemical and physical characteristics, media can be distinguished qualitatively as: Solid vs broth Non-synthetic vs chemically defined Reducing Selective Differential 3Lecturer: Mr M. Zivuku

Definitions Solid medium [agar] Solid medium is media containing agar or some other, mostly inert solidifying agent. Solid medium has physical structure (broth lacks structure) and this allows bacteria to grow in physically informative or useful ways (e.g., as colonies or in streaks). Solid medium is usually used as: -Slants -Stabs -Petri dishes See illustration below. 4Lecturer: Mr M. Zivuku

Various examples solid media 5Lecturer: Mr M. Zivuku

Definitions Colony A colony is a pile or mass of a sufficiently large number of cells, growing on or in solid medium, that they are visible to the naked eye. Broth medium Broth medium is media lacking a solidifying matrix (see solid medium). Non-synthetic [chemically undefined] medium Chemically undefined ingredient: –Non-synthetic medium contains at least one component that is neither purified nor completely characterized nor even completely consistent from batch to batch. –Often these are partially digested proteins from various organismal sources. 6Lecturer: Mr M. Zivuku

-Nutrient broth, for example, is derived from cultures of yeasts. -May be broth or solid. 7Lecturer: Mr M. Zivuku

Pure culture technique Clonal populations: –Pure culture technique is a method of culturing microorganisms in which all of the individuals in a culture have descended from a single individual. –This is done so as to: inhibit evolutionary change within cultures allow the characterization of types microorganisms without the confounding presence of other, different types of microorganisms 8Lecturer: Mr M. Zivuku

Pure culture technique Colony isolation: The basis of pure culture technique is the isolation, in colonies, of individual cells, and their descendants, from other colonies of individuals. This is usually done by culturing methods employing Petri dishes such as: –Streaking –Pouring –spreading 9Lecturer: Mr M. Zivuku

Pure culture techniques Isolation from the wild: When isolating microorganisms from complex mixtures it is always a good idea to repeat the isolation procedure at least once (e.g., restreak an isolated colony) to make sure that an isolated colony is truly derived from only a single cell (i.e., closely overlapping colonies can be indistinguishable from colonies founded from single cells) Following their isolation from the wild, microorganisms may be characterized by inoculation into differential medium to determine what type of nutrients they require or can use, and what types of by-products they produce. This aids in identification 10Lecturer: Mr M. Zivuku

Colony morphology Differentiating colonies: –Colony morphology gives important clues as to the identity of their constituent microorganisms. –Important classes of characteristics include: size type of margin colony elevation colony texture light transmission colony pigmentation 11Lecturer: Mr M. Zivuku

Illustration, variation in colony margins 12Lecturer: Mr M. Zivuku

Streaking Petri dish Petri dishes are circular, vertical sided plates used to contain agar and with tops for aseptic purposes. Loop A platinum wire formed into a loop is heated to an orange glow to sterilize it then is used to transfer a culture from one physical location to another. Streaking is a method of applying cultures to solid medium: a sterile loop is cooled and brought into contact with a culture the loop is then brought into contact with the surface of solid medium whereupon it is streaked (i.e., dragged) along the surface of the solid medium colonies grow along the points of the streak 13Lecturer: Mr M. Zivuku

Illustration streaking a plate 14Lecturer: Mr M. Zivuku

Each species of bacteria produces a distinctive colony appearance. Purpose of streaking: To obtain pure, isolated colonies. Principle: By spreading a large amount of bacteria over the large surface area of a plate, the amount of bacteria is diluted until individual cells are spread on the surface of the plate. Each individual cell grows into a single colony. All of the cells in this colony are genetically identical. 15Lecturer: Mr M. Zivuku

Streak pattern Bacterial growth pattern Nice isolation! 16Lecturer: Mr M. Zivuku

Starting with a mixed culture, isolation of different species may be obtained by streaking 17Lecturer: Mr M. Zivuku

Example of a poorly streaked plate: Bacterial growth is too heavy and isolation is poor. 18Lecturer: Mr M. Zivuku

Contamination of a streak plate results from leaving the plate open too long or not shielding properly with the lid. Correct procedure 19Lecturer: Mr M. Zivuku

Which streak plate culture started as a pure culture. How can you tell? Answer: the one on the right, because all colonies look alike. 20Lecturer: Mr M. Zivuku

Pour plate Method A pour plate is a method of melted agar inoculation followed by petri dish incubation. Steps include: –Cultures are inoculated into melted agar that has been cooled to 45 � C –the liquid medium is well mixed then poured into a petri dish (or vice versa) –colonies form within the agar matrix rather than on top as they do when streaking plate Pour plates are useful for quantifying microorganisms that grow in solid medium. 21Lecturer: Mr M. Zivuku

Pour plate method Because the "pour plate" embeds colonies in agar it can supply a sufficiently oxygen deficient environment that it can allow the growth and quantification of microaerophiles Spreading a plate is an additional method of quantifying microorganisms on solid medium. –Instead of embedding microorganisms into agar, as is done with the pour plate method, liquid cultures are spread on the agar surface using a devise that looks more or less like a hockey stick. An advantage of spreading a plate over the pour plate method is that cultures are never exposed to 45 � C+ melted agar temperatures. 22Lecturer: Mr M. Zivuku

Preservation of microbes Methods of preserving cultures include: Refrigeration Stabs Slants Lyopholisation freezing 23Lecturer: Mr M. Zivuku

24 Aseptically pouring agar plates

Lecturer: Mr M. Zivuku25 Agar plates are stored upside down to prevent condensation.

Lecturer: Mr M. Zivuku26 1.Initials 2. Date (mm/dd/yy) 3. Code # or letter All labeling is done on the bottom of the agar plate

Lecturer: Mr M. Zivuku27 Environmental sampling

Lecturer: Mr M. Zivuku28 Typical environmental sampling results

Lecturer: Mr M. Zivuku29 Microbes in the Environment. This plate was inoculated with 500 L of indoor air. Approximately how many colonies are on the plate? How many different bacterial species are here?

Lecturer: Mr M. Zivuku30 Microbes in the Environment. This plate was inoculated with a cotton swab that was wiped over a shower drain. Fungi usually produce large, "fuzzy" colonies (marked F). Approximately how many colonies are on the plate? How many are bacterial colonies? How many different bacterial species are here?