CHAPTER 3 - Isolation & Cultivation of Microorganisms

______________ - a culture which contains a single species of organisms; a population of cells arising from a _________ cell

pure culture (axenic culture), single

________________ - increasing the population of microorganisms by providing their nutritional and physical requirements

cultivation

___________ - extracellular substances which provide the cell with materials for building protoplasm and for energy generation

nutrients

__________________ - any nutrient material for growth and cultivation of microorganisms in the laboratory

culture medium

uses of culture medium

• for growth and maintenance of microbial cultures

• to favor the production of particular compounds

• to study microbial action on some constituents of the medium

types of culture media, according to physical state:

• liquid (broth)

• semi-solid

• solid

what is the solidifying agent used in a culture media?

agar/gelatin

types of culture media, according to physical state:

• ____________ - no solidifying agent

• ____________ - 0.1 - 0.5% solidifying agent

• _____________ - 1.5 - 2.0% solidifying agent

liquid (broth), semi-solid, solid

liquid and solid types of culture media

if you want to see motility of a microorganism, what type of culture media should u use?

semi-solid

types of culture media according to chemical composition:

• synthetic

• complex

a type of culture media in which all components are chemically defined.

synthetic

a type of culture media in which no all components are chemically defined

complex

examples of complex culture media (according to chemical composition)

• potato infusion (plant origin)

• beef extract (animal origin)

• yeast extract (microbial origin)

Types of culture media according to principal function, purpose or application

• General purpose

• Differential

• Selective

• Enrichment

• Assay

a type of culture media that can support most or all types of species; with the use of Nutrient Agar (NA) and Potato Dextrose Agar (PDA)

General Purpose

a type of culture media that is used for enumeration of molds

Potato Dextrose Agar

a type of culture media that distinguishes one type of bacteria from another; with the use of special reagents like __________

Differential, pH indicators/dyes

An example of a pH indicator/dye

Eosin Methylene Blue Agar (EMBA)

Eosin Methylene Blue Agar (EMBA)

on NA = normal

on EMBA = with dyes that can inhibit gram-positiv

_____________ - a selective medium to separate vibrio species; very alkaline; yellow color means _________

Thiosulphate-citrate bile salt

CHROMagar Candida medium - different shades of blue/green color

a type of culture media that allows the growth of a specific type of microorganism only; with ______________ agents (ex. salts, dyes, antibiotics)

Selective, selective

It is an example of selective culture media - _______________

Bacillus Cereus Agar; blue-green in color with white precipitates

a type of culture media that is used to increase the number of microorganisms with unusual physiological characteristics; with special nutrients (_______). Give an example of medium used

enrichment, Blood Agar

_____________ - useful in classifying streptococcal species; a substance that causes hemolysis is a ____________

Blood agar, hemolysin

________Hemolysis - complete

_________ - partial only

_________ - non hemolytic

Beta, Alpha, Gamma

_______________ - breaks down blood

Hemolysis

a type of culture media used for investigative study for quantitative/qualitative data; of prescribed composition of used _______ of vitamins, amino acids and antibiotics.

assay, assay

a type of culture media that is used to determine qualitative/quantitative production of such a compound by an organisms

assay

_____________ -a type of culture media (assay) that uses three sugars: _______, ________, and _____________

Triple sugar iron medium

glucose, sucrose, and lactose

it utilizes glucose as it changes to color yellow

another example of assay that is used for antibiotic testing

Disk diffusion assay or Kirby-Bauer test

Enumerate the isolation techniques

• plating

• enrichment culture

• serial dilution

• single-cell isolation technique

• membrane filter technique

______________ - a macroscopically visible (surface/subsurface) growth of cluster of microorganisms on a solid medium

colony

different types of plating

• streak

• spread

• pour

types of plating and its corresponding place of growth

____________ - surface agar

____________ - surface agar
_____________ - subsurface/surface agar

• streak

• spread

• pour

__________ - can be seen in the surface only; most commonly used

streak plating

a type of plating technique that is spread evenly over surface of agar using sterile glass spreader

spread-plate technique

a plating technique that uses a sterile medium and is mixed well with inoculum

pour-plate technique

an isolation technique that isolates specific types of microorganisms by a combination of nutrient and physical conditions

enrichment culture

______________ - used for the isolation of unusual physiological types of microorganisms which are present in small numbers and which grow slowly

enrichment culture

_________________ - used if the desired microorganism is present at higher level than any other microorganism

serial dilution

______________ - used this type of isolation technique when studying organisms from the soil

serial dilution (coupled technique: serial dilution + plating)

__________ uses a micropipette or microprobe to physically pick a single cell and transfer it on an agar medium

Single-cell isolation technique

different single-cell isolation techniques

_______________ - an isolation technique that is used for samples with low population

Membrane Filter Technique

Steps in preparing pure cultures (for preservation)

• isolation

• transfer the desired colony to a slant/stab

• Verify the purity (pure culture, check if gram (+) or gram (-)

  • microscope

  • restreak on agar medium (check phenotype if still the same)

  • physiological and biochemical test (normally: rapid kit)

• Make stock cultures

the objective of culture preservation: to retain the ___________ of the stock culture for a ______ period of time while maintaining its _________

viability, long, purity

Culture preservation methods

• periodic transfer to fresh media

• overlaying cultures with mineral oil

• freeze drying (lyophilization)

• freezing with liquid nitrogen

• drying

periodic transfer of fresh media (sub culture); considerations:

• time _________ of transfers

• proper______

• proper storage ______

• interval (has limitations; 4-5 transfers)

• medium

• temperature

in this culture preservation method it limits the availability of oxygen

Overlaying cultures with mineral oil

what does limiting the availability of oxygen do in a culture?

it reduces the metabolic rate of the culture

Some reminders in overlaying cultures with mineral oil

• allow it to grow (culture) before placing the mineral oil

• after allowing it to grow, proceed to overlay with mineral oil

• place in a refrigerator

Advantages of overlaying cultures with mineral oil

• prevents ____________

• simple

• enables one to remove some __________ under the oil and inoculate it in a _________ medium and still preserve the initial culture

• dehydration

• growth, fresh

disadvantages of overlaying culture with mineral oil:

• ___________ of microorganisms varies with species

• not all possible to be stored

viability

A culture preservation method that involves rapid drying in frozen state (temp: -20°C)

lyophilization/freeze-drying

process of freeze-drying

freeze dry until H20 molecules are removed for preservation and needs a vacuum pumpad

advantages of freeze-drying (lyophilization)

• _______- term survival

• _________ opportunity for changes in the characteristics of culture

• _________ storage containers

• long

• less

• small

a culture preservation method that uses temperature around -196°C (cryopreservation)

freezing with liquid nitrogen

considerations for freezing with liquid nitrogen

• cryoprotective agent (______)

• liquid-nitrogen refrigerators

  • pulverized in form

  • exposed to ice crystals; reason for protecting it with _________

glycerol - cuz coating siya

a culture preservation method (____________) - in which samples are grown on sterile paper disk saturated with nutrient, then the disks are allowed to air dry and stored aseptically, drying temperature = 45°C

drying

limitations of drying as a culture preservation method

only used for spore- and cyst- formers

______________ organizations which maintain authentic pure cultures of microorganisms

culture collection

______________ provides “________” (first strain isolated from a species) strains to microbiologists

culture collection, type