Post Lab Pure Culture Isolation Flashcards
is the process of preserving the integrity and functionality of cells, tissues, and organs held
outside the native environment for extended storage times
biopreservation
The primary aim of culture preservation is to
maintain
the organism alive, uncontaminated, and without variation
or mutation, that is, to preserve the culture in a condition
that is as close as possible to the original isolate
in fact, some strains of the same species give ____results
with the same procedure
variable
It should be emphasized that the
success of any preservation method depends on the use of
the appropriate medium and cultivation procedure and on
the age of the culture at the time of preservation. This is
particularly true when working with bacteria that contain
___ or ____ ___or that exhibit growth
phases such as morphogenesis or spore formation
plasmids
recombinant DNA
It should be emphasized that the
success of any preservation method depends on the use of
the appropriate medium and cultivation procedure and on
the age of the culture at the time of preservation. This is
particularly true when working with bacteria that contain
plasmids or recombinant DNA or that exhibit growth
phases such as ____or ________
morphogenesis
spore formation
is an important and often neglected aspect of stock culture maintenance. Too often, isolates are
not given strain designations or, if they are, the designations
are not documented in strain data files
record keeping
) refers to a specific strain with distinct phenotypic characteristics which should be recorded
strain designation
The traditional method of preserving bacterial cultures is by
periodic serial transfer to fresh medium
The major disadvantages of the ____ technique are the risks of
contamination, transposition of strain numbers or designations (mislabeling), selection of variants or mutants, and
possible loss of culture, as well as the required storage space.
serial transfer technique
Three conditions must be determined when using this
method for the preservation of cultures:
serial culture technique
uitable maintenance medium
ideal storage temperature
frequency
between transfers
is not recommended as a longterm preservation method.
subculturing
are preferred for subculturing because they
lower the metabolic rate of the organism and thus prolong the period between transfers.
minimal media
When a complex
medium is used, more ____transfers may be necessary as
a result of accelerated growth or metabolite accumulation
frequent
A variety of bacteria, including members of the Archaea
(11, 19, 21, 29), have been preserved by subculturing in
biphasic (liquid-solid) medium
is the preferred method
for subculture is storage in a ___
refrigerator
; however, if refrigeration presents a problem,
the simplest method is storage at r
room temperature in a test tube rack or specially constructed box containing shelves with holes
Cultures stored in this
fashion require constant care since they tend to dry quickly
and, unless the laboratory has a controlled environment,
are subject to ___fluctuations.
temperature
To minimize dehydration, use screw caps with rubber liners, wrap the tube caps in ____
parafilm
Do not
select single colonies in transferring cultures, since the chances
of selecting a mutant are greater when this technique is
used.
what preservation method
serial transfer
Many bacterial species can be successfully preserved for
months (or years) simply by immersing in a ___
sterile medicinal grade oil
type of oil with a specific gravity of
0.865 to 0.890 is also satisfactory (16).
paraffin oil
Sterilize 5 ml of oil per Pyrex test tube (18 by 150 mm),
sealed with a metal or other heat-resistant closure by what sterilization technique
dry heat
Contamination of cultures when this method is used is often due
to improperly sterilized mineral oil. Each lot of sterilized oil
must be tested for sterility by streaking on an appropriate
medium such as
nutrient agar
trypticase soy agar (TSA)
After growth reaches late
___ phase or ____occurs, add the sterile mineral oil aseptically to a depth of at least 2 cm (a slant must
be entirely covered) to prevent dehydration and to reduce
metabolic activity and growth of the culture (16, 33).
logarithmic
sporulation
Store the oil-covered culture upright in a refrigerator.
Perform viability tests periodically by
removing a loopful of
growth and touching it to a sterile filter paper strip to remove the oil
Streak the growth on agar plates containing a
suitable growth medium such as nutrient agar or Trypticase
soy agar.
Confluent growth represents good survival, but plates
which show few colonies indicate a low viability, and new
stocks must be prepared
Not recommended due to cell damage from this process.
ordinary freezing
cryoptorective agents like __ or ___ can help
DMSO
glycerol
Control rate of cooling to avoid cell damage
what rate
1 to 10oC/min
Superior method at -70°C in a mechanical freezer with cryoprotective agents.
what method
deep freezing
Preserve bacterial suspension with what percent glycerol for deep freezing
30-50
Use ____ for additional preservation (15% glycerol, vials at -70°C) in deep freezing
glass beads
Long-term storage by ____bacteria, especially effective for spore-forming bacteria.
drying
in drying method, spores are resistant due to dehydrated ___
protoplast
Inexpensive method using sterile filter paper strips or disks.
what method
paper method
Suitable for quality control cultures.
paper method
Store disks in tubes, use sterile forceps to inoculate broth when needed.
what method
papermethod
Preserve ___bacteria in dried gelatin drops or disks.
heterotrophic
what temperature is better for gelatin method
-20°C
what is used for gelatin method with dessicator
phosphorus pentoxide.
To propagate, transfer a ___ drop into suitable medium.
gelatin methofd
gelatin
Preserve bacteria by drying on sterile silica gel granules.
what method
silica gel method
Preserve bacteria by drying under vacuum on perforated glass beads or porous porcelain beads.
what method
porous glass and porcelain beads method
Suitable for long-term preservation of bacteria and bacteriophages.
freeze-drying (lyophilization)
lyophilization is suitable for long-term preservation of
bacteria
bacteriophages
Removes water from frozen bacterial suspensions by sublimation under reduced pressure.
lyophilization
Dried cells stored away from oxygen, moisture, and light. Rehydration restores them.
what method
lyophilization
system of lyophilization includes a vacuum pump, condenser, thermocouple vacuum gauge, and heavy-walled tubing.
simple system
Centrifuge cell suspension, freeze by vacuum evaporation, constrict vials, and secondary freeze-dry.
what method of lyophilization
centrifugal freeze-drying
More frequently used. Simple lyophilization system with high-vacuum pump and condenser.
prefreezing method
Easier to handle, less contamination risk.
Outer vials contain silica gel granules and cotton wad.
Inner vials contain bacterial suspension.
Labeling: Use ink that withstands ultralow temperatures and solvents. Labeling machine recommended.
what vial
double vial
Used when sucrose is the cryoprotective agent.
what vial in lyophilzation
bulb shaped vial
Used when the culture is suspended in skim milk.
what vial in lyophilization
tubular vials
examples of cryoprotective agents aside from glycerol or DMSO
Skim milk, sucrose, dextran, horse serum, inositol, raffinose, trehalose, methylcellulose, glycine betaine, charcoal.
Preparation:
Prepare a 20% skim milk solution, sterilize at 116°C for 20 minutes.
For agar cultures: harvest growth with skim milk solution.
For broth cultures: harvest by aseptic centrifugation, resuspend pellet in skim milk to 108 cells/ml.
Standardize cell concentration using OD (0.1 = 108 cells/ml).
Use sterile 20% skim milk, avoid for bacteria inhibited by milk (use sucrose instead).
Dispense 0.2 ml of cell suspension into each vial, trim cotton plugs.
what metho
double-vial chamber method
Preparation:
Dispense 0.2 ml bacterial suspension into each sterile vial.
Push cotton plug 1.3 cm below vial rim, flame rim to remove fibers.
Attach sterile latex IV tubing to vial rim using tube stretcher.
Freeze-Drying Process:
For tubular vials: freeze in dry-ice–ethylene glycol bath, attach to manifold.
For bulb-type vials: shell-freeze suspension, attach to manifold.
Dry at vacuum <30 µm Hg for 18 hours.
Post-Drying:
Seal vials below cotton plug with dual-tipped air/gas torch, moving flame up and down.
Allow vials to cool.
what method
double vial chamber method
Preservation in liquid nitrogen at -196°C or vapor phase at -150°C.
Successful long-term preservation of fastidious bacteria for over 30 years.
Ultralow-temperature mechanical freezers at -70°C can also be used.
Precautions against electrical shutdowns or compressor malfunction; use alarms, backup freezers, or generators.
what method of preservation
ultrafreezing
Glycerol and DMSO, pass through the cell membrane.
what kind of cryoprotective agent
intracellular and extracellular protection
Dextran, glucose, lactose, mannitol, polyglycol, polyvinylpyrrolidone, sorbitol, sucrose.
what kind of cryoprotective agent
extracellular protection
are equally effective for a wide range of bacteria.
glycerol
DMSO
Use actively growing cells at mid-___to late ___ phase.
logarithmic
Harvest by aseptic centrifugation, resuspend pellet in sterile fresh medium with 10% glycerol or 5% DMSO.
what culture prep method
broth
Wash growth from agar surface with sterile broth containing cryoprotective agent.
what culture prep method
agar
Cooling Rate: Slow cooling (1°C/min) for best preservation.
Procedures:
Programmable Freezer: Freeze at 1°C/min to -40°C, then at 10°C/min to -90°C.
Manual Freezing: Stainless steel pan in mechanical freezer at -60°C for 1 hour, then liquid nitrogen bath for 5 minutes.
Alternative: Sealed vials in 95% ethanol in mechanical freezer set at -85°C, then transfer to liquid nitrogen.
what method
freezing
: Greatest recovery of bacteria.
rapid thawing
Consult____ for maintenance and preservation of bacterial genera.
Bergey’s Manual of Systematic Bacteriology
Maintain strictly __conditions during growth, harvesting, dispensing, and freezing.
for anaerobes
anaerobic
Use ___ cryoprotective agents for anaerobes
prereduced
Grow in prereduced broth in Hungate test tubes, harvest cells by centrifugation
what genera
anaerobes
Flush pipettes, syringes, and vials with oxygen-free gas.
what genera
anaerobes
Freeze-drying cryoprotectant: ____ (Trypticase soy broth, sucrose, bovine serum albumin, distilled water) for anaerobes
reagent 18
Freeze in dry-ice–ethylene glycol bath or store in liquid nitrogen.
what genera
anaerboes
Preserve by freezing and storage in liquid-nitrogen vapor.
Cryoprotectant: DMSO (5% vol/vol) in maintenance broth.
what genera
cyanobacteria
Harvest cells at early ____ phase, resuspend in cryoprotectant ___ (bovine serum albumin, sucrose, distilled water) for cyanobacteria
stationary
reagent 20
Require stringent anaerobic conditions for growth.
Preserve by freezing and storage at low temperature.
Grow on H2-CO2 gas mixture in heavy glass-walled test tubes or serum bottles.
Harvest cells, resuspend in medium with 10% glycerol or 5% DMSO.
Use N2-CO2 gas mixture for flushing vials.
Freeze and store in vapor phase of liquid-nitrogen container.
what genera
methanogens
Sensitive to preservation process at different growth stages.
Harvest cells at proper growth stage for preservation.
what genera
Neisseria, Haemophilus, Campylobacter, and Helicobacter Species:
Preservation depends on plasmid stability.
Stable plasmids can be freeze-dried or frozen by standard methods.
Enhance recovery of unstable plasmids by growing in antibiotic-containing medium before and after preservation.
what genera
plasmid-containing bacteria
can be freeze-dried or frozen by standard methods.
what kind of plasmids
stable plastmids
Enhance recovery of ___plasmids by growing in antibiotic-containing medium before and after preservation.
unstable
Preserve using sterile, air-dried soil.
Sterilize soil by autoclaving, inoculate with spore suspension, dry, and store in refrigerator.
what genera
sporeformers
Valuable resources for microbial germplasm.
Serve specific research aims or house diverse microorganisms, cell lines, viruses, and genetic material.
culture collections
three types of culture collections
specialized
reference
national
Personal and not intended to be permanent.
Narrow variety of microorganisms but extensive in strains.
Indispensable for studies in taxonomy, genetics, and strain variability.
what kind of culture collection
specialized collection
example of specialized collection
Mortimer Starr’s bacterial plant pathogen collection at UC Davis.
Permanent national resources like ATCC (USA) and NCTC (England).
Main functions: Acquire, authenticate, preserve, and distribute reference and type cultures.
Provide continuity with past microbial strains for taxonomic research and biotechnological applications.
Broad variety of species but may have fewer strains within each species.
national collection
Serve as national and international patent depositories.
Offer services such as strain identification, safe deposit, and specialized training.
Source of strain information, history, data, and applications.
what culture collection
national collection
are involved in collaborative efforts
of scientists, industry, academe, and government agencies
and are concerned mainly with one major group or function. These collections may be located in government agencies, universities, or industries associated with the production of goods, chemicals, and drugs
reference collection
natural microbial populations are ___ cultures
mixe
mass of growth of cells containing only one type of cells is called __
pure culture
selective methods include
chemical
physical
biology
chemical methods of selection include
enrichment selection (special carbon or nitrogen)
dilute media
use of enrichment medium containing ___ to isolate bacteria which can degrade this
a-conidendrin
use of enrichment medium containing single source of nitrogen ___ for nitrogen fixing bacteria
N2 gas
use of dilute media for isolation (0.01% peptone) is for ___
caulobacter spp.
isolation of gram negative bacteria includes the addition of ___ in MacConkey’s Agar
bile salts or crystal violet
vancomycin, polymyxin, trimetrophim is added to medium to inhibit other interfering intestinal bacteria to isolate this
campylobacter jejuni
physical methods of selection includes
heat treatment
incubation tempterature
pH
cell size and motility
heat treatment of 80oC for 10 mins is for ____
endospore producing bacteria
incubation temp for thermophiles
55oC
incubation temp for psychrophiles
0-5oC
isolation of lactobacilli, set pH to ___ with ___ buffer to maintain pH
5.35
acetate buffer
isolation of vibriae cholera set pH to ___
8.5
isolation of __ from oral cavity include the use of membrane filter with pore size of 0.15um
treponema spp.
treponema ___ through the filter on underlying agar medium
passes
example of biological method of selection
use of susceptible laboratory animals
isolation of ___ from sputum sample, injecting sputum sample of patient in mice
s. pneumoniae
methods of isolating pure culture (4)
streak plate
pour plate
spread plate
micromanipulator
dilution of bacteria on solid media, done by streaking the culture over agar surface
streak plate technique
pattern of streaking using streak plate
four quadrant method
bacteria get separated from each other by sufficient distance
streak plate
one organism produces one colony and is considered as a ___ cutlure
pure
colonies developed after streak plate method may require repeated ___for isolation of bacteria from mixed culture
streaking
used for isolation of anaerobic bacteria
roll tube technique
advantages of streak plate technique
less material required
less laborious
surface colonies
modification of streak plate technique for isolation of anaerobic bacteria
roll tube technique
stoppered anareobic culture tobes whose inner walls are coated with ___ media are used, tubes are filled with ___ nitrogen
pre-reduced
oxygen free nitrogen
to keep anaerobic bacteria from oxygen exposure after removal of stopper, they are flushed with oxgen free __ from ___
CO2
gas cannula
inoculation done with transfer loop in anaerobic bacteria held against agar surface as tube being rotated by a ___
motor
inoculation of ___bacteria starts at the bottom and drawing the loop gradually upward, the inoculum becomes thinned and well isolated colonies are obtained
anaerobic
serial dilutions added to sterile sufficiently cooled agar medium and mixed
pour plate technique
advantage of pour plate method
method can be used to determine number of bacteria in sample, quantitative method
disadvantage of pour plate
laborious
more material
time consuming
some of the colonies are submerged
exposure to 45oC (cannot be used to isolate psychrophiles)
serial dilution of mixed culture added onto surface of agar plate, spread evenly
spread plate
advantage of spread plate
surface colonies
no exposure to higher temp
quantitative
use instrument and microscope in conjunction
micromanipulator
allows controlled movement of micropipette
micromanipulator
picked up with micropipette while observing through microscope
single cell
single cell is added to nutrient medium, after incubation pure culture is developed from a single cell
what method
micromanipulator
in micromanipulator, the culture is a ___
clone
