Chapter 3: Tools for the Lab: Methods of studying/culturing microorganisms

What are the Five I's?

culturing microbes(techniques to manipulate, grow, examine, characterize): inoculation, incubation, isolation, inspection, identification

Inoculation

producing a culture by introducing microbes into or upon media for culture

what is required for inoculation

sterile tools

culture

propagation/growth of microorganisms in or on a nutrient medium

medium

a nutrient

sterile

free of all life forms including spores and viruses

Incubation

once a container of medium has been inoculated with a specimen, it is placed in a temp-controlled chamber (incubator) to encourage growth

incubation temp

20 to 45 degrees Celsius

microbial growth in liquid medium

cloudiness/turbid, sediment, scum, color/pigment

microbial growth on soli medium

colonies

3 properties of media classification

physical state

chemical content

function

physical state

liquid (broth, milks, infusions)

semisolid (contains gelatin/agar)

solid (can be liquified)

solid (can't be)

semisolid media function

used to examine motility of bacteria, provides backdrop

chemical comp

chemically defined (synthetic)

complex & not chem defined

functional type

many (8)

general purpose media

designed to grow as many diff types of microbes as possilble

enriched media

contains complex organic substances

fastidious

bacteria that require growth factors and complex nutrients

selective media

speed up isolation by suppressing unwanted organisms & favoring growth of desired ones

differential media

Isolation

separating one species from another (allows the growth of colonies)

colony

a discrete mound of cells

how to isolate?

petri dish x inoculating tools

3 isolation methods

streak plate, pour plate, spread plate

streak plate method

small drop of culture is spread across surface of a medium with I-loop

-patterns used for separation for discrete colony growth

pour plate

-sample is diluted into cooled but still liquid agar tubes

-tubes r poured into sterile petri dish, allowed to solidify

-some colonies may grow embedded in medium rather than just on the surface

spread plate

small amount of sample is pipetted onto surface of plate

-sterile spreading tool used

pure culture

consists of only one species of microorganisms

anexic

free of other living things except for the one being studied

subculture

a second level culture from a well-isolated colony

mixed culture

container holding 2 or more identified species of microbes

contaminated culture

once pure or mixed but now contains contaminants

contaminants

unwanted microbes of uncertain identity

Inspection

helps determine what sorts of microbes have been isolated in cultures

what doe inspection entail?

-observing colony morphology and -color

-examining growth inside broth tubes

-microscope

what does identification entail?

biochemical tests- technique to identify bacteria

what identifies microbial profiles?

-phenotypic, genotypic, immunologic testing

-macro & microscopic analysis

microscope characteristics

microscope types

what does the method of specimen prep depend on?

-whether specimen is in living or prepared state

-the goals of the observer

-type of microscope available

staining techniques

simple, differential, special

simple stain

use a single base dye solution, all cells appear the same color

-reveals shape, size, arrangement

-types: malachite green, crystal violet, mordant

differential stain

uses 2 diff dye solutions, primary, & counterstain, to distinguish btw cell types (bacteria) or parts

-types: gram, acid-fast, endospore

special stain

used to emphasize or identify certain cell structures not seen in conventional staining

-types: ,bncapsule, flagellar