Lab Technique 1: Media Preparation and Sterilization

culture medium

a solution that contains nutrients, such as carbon and nitrogen, required for cells to grow, agar can be added to made solid

complex medium

composed of digests of chemically undefined substances such as yeast and meat extracts or digests

defined medium

one for which a precise chemical composition is known

T/F: Nutrient agar and Trypticase Soy Agar are complex, general purpose media for the isolation and cultivation of a variety of microorganisms

true

T/F: Sabouraud Dextrose Agar is a defined medium used when cultivating fungi or yeasts.

false. Sabouraud Dextrose Agar is a complex medium used when cultivating fungi or yeasts

what is liquid medium called?

broth

what can broths be in?

• test tube (1-10mL)

• flasks (10mL-2L)

• carboys (4-25L)

• or a larger vat

what is the solidifying agent in solid media?

usually agar which is extracted from Rhodophyta—red algae

why is agar extracted from Rhodophyta—red algae a useful solidifying agent?

it melts at 100^oC and solidifies at 40-42^oC and unlike other solidifying agents like gelatin, it is not easily broken down or digested by microbes

T/F: agar media can only be used in petri dishes

false. it can be in tubes or petri dishes

plates

solid media in petri dishes; the surface can be used for the isolation and growth of microorganisms

deeps

test stubes containing solid medium, which has been allowed to solidify in an upright position, and are usually used for anaerobic growth

slants

test tubes containing solid medium that has been allowed to solidify at an angle and the surface of the agar is used for the maintenance of stock cultures of microorganisms

T/F: media can be selective, differential, both or neither.

true

selective medium

a nutrient medium designed to favor the growth of certain microbes and to inhibit undesirable competitors

differential medium

a medium which provides a visible indication of physiological characteristic of a microorganism

T/F: Carbohydrate fermentation or enzyme production could be demonstrated on selective media

false. Carbohydrate fermentation or enzyme production could be demonstrated on differential media

Phenyl ethyl alcohol agar (PEA) is…

selective only; it selects for Gram-positive organisms but tells nothing of the physicology of the organism growing on it

Mannitol salt agar (MSA) is …

both selective and differential; it selects for growth of salt-tolerant organisms and differentiates mannitol fermenter (turn from red to yellow) from non-mannitol fermenters (don’t change color of agar)

Eosin methylene blue agar (EMB) is…

both selective and differential; it selects for Gram-negative bacteria and differentiates lactose fermenters (colonies take up dye) from non-lactose fermenters (colonies are colorless; no dye uptake)

sterilization

the killing or removal of all living organisms and their viruses from a growth medium

autoclave

the most common form of sterilization; it is a sealed device that allows the entrance of steam under pressure delivering moist heat kills all microorganisms including bacterial endospores (which are extremely heat-resistant)

what temperature and pressure is the autoclave? for how long?

121^oC at 15 pounds per square inch (psi) pressure for 15 minutes-1hr

what is autoclaving mainly used to sterilize?

media and glass

dry heat sterilization

kills by oxidation effects; direct flaming

dry heat usually sterilizes..

inoculating loops, needles, glass

hot-air sterilization

the use of an oven heated to 170^oC for 2 hours for sterilization of glassware

filtration

a method used for heat-sensitive liquids (like antibiotics) that has pores too small for the passage of microorganisms but large enough for the liquid or gas to flow through

gas chemosterilizers (ethylene oxide)

devices that use gas like ethylene oxide to sterilize (cold-sterilization) heat-sensitive objects such as Petri dishes

besides petri dishes what can ethylene oxide also sterilize?

• paper

• leather

• wood

• metal

• rubber

electromagnetic radiation

microwaves, ultraviolet radiation, x-rays, gamma rays, and electrons used to sterilize

Ultraviolet (UV) radiation

electromagnetic radiation with wavelength between 10nm and 400nm, shorter than visible light but no longer than lethal x-rays; causes damage to the DNA (via thymine dimers) leading to the death of the exposed organisms unable to adequately repair

Ionizing radiation

causes ions and other reactive molecules to be produced. these reactive molecules degrade and alter DNA and proteins

electromagnetic and UV radiation can sterilize…

does not penetrate solids but can be used to sterilize exposed surfaces of thin layers of solution

T/F: radiation can be used in the food industry and for sterilization and decontamination of medical supplies

true