Preparation of Culture Media, Inoculation & Incubation Techniques Flashcards
Increase in cell number through binary fission
Microbial growth
cell simply splits into two cells, producing two identical cells
Binary fission
Difference of binary fission from mitosis
No mitotic spindle form
the average time between two consecutive generations in the lineages of a population
generation time
generation time E.coli
30 to 180 mins
generation time M. tuberculosis
12 hours
T. pallidum
33 hours in rabbit testes
M. leprae
10 days in armadillos
Cocci synthesize new cell walls
septal region
Synthesize new cell wall entire surface of the cell
Rod shape bacteria
Logarithmic representation of bacterial growth
2^n
cell form an initial outgrowth that enlarges until its size approaches that of a parent cell and then it separates
Budding
the initial outgrowth
a bud
microbes that grow and multiply in or on a culture medium
culture
A group of descendants of an original cell
colony
contains a single microbial species
pure culture
other name of pure culture
axenic culture
how the surface of the colony appears
texture
Texture types
smooth
glistening
mucoid
slimy
dry
powdery
flaky
Transparency
Transparent
Translucent
Opaque
You can see through them
Transparent
Light pass through them
Translucent
solid-appearing
opaque
Intracellular pigments
color/pigmentation
Many bacteria do not produce any pigment and appear __________
white or gray
Magnifying tool
Colony counter
Dissecting microscope
Colony size
Punctiform <1 mm
Small 1-2 mm
Medium 3-4 mm
Large >5 mm
Colony forms
Circular
Rhizoid
Irregular
Filamentous
Colony Margin
Entire
Undulate
Lobate
Filamentous
Curled
Colony elevation
Flat
Raised
Convex
Pulvinate
Umbonate
Rely on organic compounds as source of food to provide energy and carbon
Heterotrophs
Most lab bacterias we work with
Mesosphile
Mesophile/Mesophilic temperature
20 to 40 degrees celsius
Body Temperature
37
Room temperature
25
The lowest temperature at which an organism is able to conduct metabolism
Minimum growth temperature
Temperature at which an organism’s metabolic activities produce the highest growth rate
optimum growth temperature
The highest temperature at which an organism continues to metabolize
maximum growth temperature
when temperature exceed maximum growth temperature, the organism’s _________
protein are permanently denatured and dies
P S Y C H R O P H I L E S other name
cryophiles
sensitive to temperatures over 20 ̊C
optimum growth at
15 ̊C or below
P S Y C H R O P H I L E S
− optimum growth at 20 ̊C to 30 ̊C
P S Y C H R O T R O P H S
− optimum growth at 25 ̊C to 40 ̊C
M E S O P H I L E S
− optimum growth at 50 ̊C to 60 ̊C
T H E R M O P H I L E S
P S Y C H R O T R O P H S Other name
Cold-tolerant bacteria or archaea
grow at 80 ̊C or higher
Extreme Thermophiles
M E S O P H I L E S other name
moderate temperature-loving
T H E R M O P H I L E S other name
Heat-loving
T H E R M O P H I L E S example
Bacillus stearothermophilus
Extreme Thermophiles Example
Geogemma barosii
Geogemma barosii
85 to 121
2 hours in 130
Can destroy most microbes
> 60 degrees
slow microbial growth
50 to 60
rapid microbial growth; produce toxins
25 to 50
grow at very low pH (0.1 to 5.4)
A C I D O P H I L E S
Organs with low pH
Vagina and stomach
causative agent of peptic ulcer
Helicobacter pylori
A C I D O P H I L E S example
Lactobacillus acidophilus
− grow at pH 5.4 to 8.5
N E U T R O P H I L E S
Lactobacillus produce
lactic acid
− grow at high pH (7 to 12)
A L K A L I P H I L E S
Most bacteria, protozoa, and pathogens grow best at what pH
Neutral
A L K A L I P H I L E S example
− Vibrio cholerae
cholera
− Alkaligenes faecalis
− Agrobacterium spp.
Pressure difference needed to stop the flow of solvent across a semipermeable membrane
Osmotic pressure
require very high salt conc. 20 to 30%
O B L I G A T E
H A L O P H I L E S
O B L I G A T E
H A L O P H I L E S
other name
extreme halophiles
strict halophiles
Can survive month in dry conditions due to the ability of its cell wall to retain water
Mycobacterium tuberculosis
− do not require high salt conc. for growth
− tolerates 2-15% salt concentration
F A C U L T A T I V E
H A L O P H I L E S
Salt-water principle
Water follows salt
Sodium attracts water
No net movement of water
Cells in isotonic solution
Shrink because of the net movement of water out of the cell
Cell in hypertonic solution
Undergo gain of water
Cells in hypotonic solution
Solute concentration inside and outside are the same
Isotonic solution
Have higher solute concentration than inside the cell
Hypertonic solution
Has a lower solute concentration
Hypotonic solution
Hypo comes from
Latin word under or low
− organisms which derive energy from organic carbon sources
− may need to consume other living organisms to
live
C H E M O H E T E R O T R O P H S
Organisms that obtain energy by the oxidation of electron donors in their environment
Chemotrophs
Organism that cannot manufacture its own food by carbon fixation and therefore derives its intake from other sources of organic carbon, mainly plant or animal matter
Heterotroph
− organisms which use inorganic energy sources to synthesize organic compounds
− primary carbon source is carbon dioxide
C H E M O A U T O T R O P H S
organism that can produce its own food using light, water, carbon dioxide, other chemicals
Autotroph
makes up 14% of
dry cell weight
N I T R O G E N
Nitrogen is to build __________
Proteins and nucleotides
N I T R O G E N sources
proteins,
ammonium,
nitrogen gas,
nitrates
Some bacteria can reduce nitrogen gas to
ammonia
− used to form proteins and some vitamins
S U L F U R
S U L F U R sources
proteins,
hydrogen sulfide,
sulfates
Sulfur is used to synthesize sulfur-containing amino acids and vitamins like
Thiamine - Vitamin B1
Biotin - Vitamin H
used to form DNA, RNA, ATP, and phospholipids
P H O S P H O R U S
P H O S P H O R U S sources
Inorganic
phosphate salts,
Buffers (from culture medium)
phospholipids are found in _______
cell membranes of bacteria
Growth requirement for aerobic microorganisms
O X Y G E N
O X Y G E N sources
Atmosphere,
Water
Require oxygen to live
O B L I G A T E
A E R O B E S
O B L I G A T E
A E R O B E S
example
Mycobacterium tuberculosis
Can use oxygen, but can grow in its absence
F A C U L T A T I V E
A N A E R O B E S
F A C U L T A T I V E
A N A E R O B E S
example
Escherichia coli
F A C U L T A T I V E
A N A E R O B E S
has ________ enzymes that allow toxic forms of oxygen to be neutralized
catalase
SOD (superooxide dismutase)
− Can’t use oxygen and are harmed by the
presence of toxic forms of oxygen
O B L I G A T E
A N A E R O B E S
O B L I G A T E
A N A E R O B E S
example
Clostridium tetani
Can’t use oxygen, but can grow in its absence
Tolerates presence of oxygen
A E R O T O L E R A N T
A N A E R O B E S
A E R O T O L E R A N T
A N A E R O B E S
example
Streptococcus pneumoniae
− Require oxygen at low concentrations
M I C R O -
A E R O P H I L E S
A E R O T O L E R A N T
A N A E R O B E S
also have _________
SOD
M I C R O -
A E R O P H I L E S
example
Helicobacter pylori
Campylobacter jejuni
Toxic forms of oxygen
Hydrogen peroxide
Superoxide radicals
converts superoxide to oxygen & hydrogen peroxide
Superoxide dismutase:
converts hydrogen peroxide to water & oxygen
Catalase:
breaks down hydrogen peroxide
Peroxidase:
− CO2-loving
− require increased concentration of CO2 and lower levels of O2
C A P N O P H I L E S
C A P N O P H I L E S
Example
Neisseria gonorrhoeae
Haemophilus influenzae
Growing capnophiles
Candle jar
CO2 generating packet
Trace elements
− K
− Mg
− Ca
− Fe
− Cu
− Mo
− Zn
Used for cell wall synthesis of gram positive bacteria
Calcium
BACTERIAL GROWTH CURVE
- Lag phase
- Log phase
- Stationary phase
- Death or decline phase
No increase in number of living bacterial cells
(Phase of intense metabolic activity)
- Lag phase
(1 hour to several days)
Exponential increase in number of living bacterial cells
(Period of most rapid growth)
- Log phase
plateau in number of living bacterial cells; rate of cell division and death roughly equal
- Stationary phase
exponential decrease in number of living bacterial cells
- Death or decline phase
CULTURE MEDIA Characteristics:
i. Contains appropriate nutrients
ii. Sterile
iii. Incubated at appropriate temperature
act of introducing microorganism or suspension of microorganisms into a culture medium
Inoculation
substance containing nutrients in which cells or microorganisms can be grown; may be solid, liquid or gel
CULTURE MEDIA
microbial cells added to a medium in order to start a culture
Inoculum:
nutrient material that contains a solidifying agent
SOLID MEDIA
− Allows for isolation and examination of colonies
− Difficult to transport and store
Agar Plate
Free from any microorganism
Sterile
− Inoculating needle is used to “stab” the solid medium
− For microorganisms that prefer reduced O2
Agar Deep
− Tube of solid medium at an angle for higher surface area
− For easy storage and transportation
− Use for long term maintenance of isolates
Agar Slant
CULTURE MEDIA common ingredient
Peptone
Beef extract
Yeast extract
Distilled water
agar
− Melts at above 95 ̊C; once melted, does not solidify until it reaches 40 ̊C
− Cannot be degraded by most bacteria
A G A R
The manner in which the inoculum is spread out into the cultured medium
Streaking
A G A R is the ____________ obtained from the cell walls of some species of red algae
Unbranched polysaccharide
some species of red algae
i. Gracilaria compressa
ii. Gelidium cartilagineum
iii. Gelidium amansii
nutrient material that lacks a solidifying agent
LIQUID MEDIA
− Liquid medium in a tube, bottle,
or flask
− Applicable if high concentration
of bacteria is desired
Broth
Who first used agar
Robert Koch
nutrient material that contains <1% solidifying agent
SEMISOLID MEDIA:
Typical agar concentration
1.5 to 2 %
the concentration of solid media
3%
Agar solidifies at what temperature
37
Another solidifying agent
Gellan gum
Solid media function
- Used to grow microorganism in their full physical form
- Used to prepare bacterial pure culture
- Use to isolate bacteria to study colony characteristic
Inoculating techniques
Stabbing
Stab and streak
Streaking
Irish moss (Red Algae)
Chondrus crispus
may be done in media with dilute agar concentration
motility test
useful in demonstrating motility of bacteria
SEMISOLID MEDIA:
For general purposes of
preservation, isolation, storage of
cultures and antibiotic sensitivity
tests
ALL - P U R P O S E
M E D I A
ALL - P U R P O S E
M E D I A
examples
− Peptone water
− Nutrient broth
− Nutrient agar
− Bismuth Sulfite Agar
Allows growth of desired
microorganism while suppressing
the growth of unwanted
microorganisms
S E L E C T I V E
M E D I A
S E L E C T I V E
M E D I A
example
− Saboraud’s dextrose agar
− Brilliant green agar
− Thayer-Martin agar
− Lowenstein Jensen Media
pH 5.6 discourages bacterial growth
for fungi
Saboraud’s dextrose agar
inhibits G+ bacteria and allows for the growth of G- salmonella
Brilliant green agar
for the growth of Neisseria gonorrheae
Thayer-Martin agar
Used to isolate Salmonella typhi
Bismuth Sulfite Agar
Used to isolate Mycobacterium tuberculosis
Lowenstein Jensen Media
Used for differentiating between
bacteria by using an identification
marker for a specific type of
microorganism
D I F F E R E N T I A L
M E D I A
D I F F E R E N T I A L
M E D I A
examples
− Mannitol salt agar
− MacConkey agar
− Blood agar
Mannitol salt agar
Staphyloccocus aureus -> halophile
MacConkey agar
Salmonella typhi
Uses hemolytic reaction
Used to distinguish bacteria that destroy red blood cells
Blood agar
Blood agar usually contains
sheep or horse blood
contains 7.5 % sodium chloride
Both selective and differential
has a pH indicator
Mannitol salt agar
Mannitol fermentation
pink to yellow positive
consider as complex, selective, and differential media
contains bile salt, and crystal violet that inhibits growth of g+ bacteria
use lactose
MacConkey Agar
The growth in _______ indicates _______ , while _______ does not
pink; lactose utilization; yellow
Lactose fermenters examples
Enterobacter spp
Citrobacter spp
Klebsiella spp
Lactose fermenters will produce
acid
test the ability of an organism to produce hemolysin, enzyme that damage/lyse red blood cells
Blood agar media
Blood agar media members
Staphylococcus
Streptococcus
Enterococcus
Partial hemolysis
Alpha hemolysis
Complete hemolysis
Beta hemolysis
No hemolysis
Gamma hemolysis
the destruction of red blood cells
hemolysis
Alpha hemolysis examples
Streptococcus pneumoniae
Streptococcus mutans
Beta hemolysis examples
Staphylococcus aureus
Streptococcus pyogenes
Listeria ivanovii
Gamma hemolysis examples
Escherichia coli
Klebsiella pneumoniae
Enterococcus faecalis
Favors growth of a particular
microorganism over others by
providing essential nutrients
E N R I C H M E N T
M E D I A
E N R I C H M E N T
M E D I A
examples
− Chocolate agar
− Loeffler’s medium
− Brain heart infusion agar
complex or particular nutrient requirement
fastidious organism
Chocolate agar example
Haemophilus influezae
Loeffler’s medium example
Corynebacterium diphtheriae (Corynebacteria)
Brain heart infusion agar examples
Streptococci
Pneumococci
Meningococci
Made up of lysed RBCs
Chocolate agar
Haemophilus influenzae
Pneumonia and sepsis
Prepared from pure chemical
substances of known composition
and concentration
C H E M I C A L L Y
D E F I N E D
M E D I A
C H E M I C A L L Y
D E F I N E D
M E D I A
example
− Peptone water
− Modified Korthof’s medium
− Firm agar
Exact components are difficult to
estimate because of complex
ingredients
C O M P L E X
M E D I A
C O M P L E X
M E D I A
example
− Nutrient agar
− Trypticase soy agar
− Eosin methylene blue agar
use to culture most chemoheterotrophic microorganism
C O M P L E X
M E D I A
Eosin methylene blue agar is used for
Isolation of the fecal coliforms (E.coli)
Eosin methylene blue agar contains lactose and dyes _______ and _______
Eosin and methylene blue
Microorganism that cannot grow in culture media
Obligate parasites
SPECIAL CULTURE TECHNIQUES armadillos
Mycobacterium leprae (leprosy)
SPECIAL CULTURE TECHNIQUES rabbit testicles
Treponema pallidum (syphilis)
SPECIAL CULTURE TECHNIQUES host cells only
Obligate parasites
STD causing
chlamydia
SPECIAL CULTURE TECHNIQUES hen’s egg
Influenza virus
SPECIAL CULTURE TECHNIQUES Cell culture
Mycoplasma spp
A method of isolating a culture by spreading microorganisms over the surface of
a solid culture medium.
S T R E A K
P L A T E
M E T H O D
A method of inoculating a solid nutrient medium by mixing bacteria in the melted
medium and pouring the medium into a Petri dish to solidify.
P O U R
P L A T E
M E T H O D
A plate count method in which inoculum is spread over the surface of a solid
culture medium.
S P R E A D
P L A T E
M E T H O D
