Microbial Growth Flashcards
increase in the number of cells or microbial population rather than in the size of individual cells
microbial growth
Factors Affecting Microbial Growth
- biochemical
factors (nutrition) - physical factors
- generation time
- biochemical
factors (nutrition)
– macronutrients
– micronutrients
– vitamins
- physical factors
– pH
– temperature
– oxygen concentration
– moisture
– hydrostatic pressure
– osmotic pressure
– radiation
supplying cells with chemical tools they need
to make monomers of macromolecules that mainly
comprise microbial cells
microbial nutrients
- made up of chemical elements
- extracellular substances that provide the microbial cell
with materials to
➢ build protoplasm
➢ generate energy
nutrients
microorganisms
Bacteria
Archaea
Protozoa
Virus
Algae
Fungi
nutrients required in relatively larger amounts
macronutrients
nutrients required in lesser quantities
Micronutrients
any nutrient material prepared/used for the growth and
cultivation of microorganisms in the laboratory
Culture Medium
Culture Media for?
- for the growth and maintenance of microbial cultures
- to favor the production of particular compounds
- to study microbial action on some constituents of the medium
no solidifying agent
inoculum preparation:
fermentation
nutrient broth
liquid
with 0.1- 0.5%
solidifying agent
Motility test
Sulfur Indole Motility
(SIM) Medium
semi-solid
with 1.5-2.0%
solidifying agent
Colony morphology
observation; hemolysis and
pigmentation characterization
Nutrient
Agar; Blood
Agar
solid
- complex polysaccharide (usually derived from
red algae) - used as solidifying agent for culture media in
Petri plates, slants, and deeps - no nutritive value; generally not metabolized by
microbes - not affected by growth of bacteria
- Liquefies at 100°C
- Solidifies at ~40°C
Agar
Types of Culture Media
(based on chemical composition)
Synthetic or chemically-defined
Complex
are not
chemically-defined
Nutrient Agar,
yeast extract
complex
All components are
chemically-defined
(precise nutrient
composition and
amounts)
Glucose
Inorganic Salt
Phosphate
Synthetic or chemically-defined
- uses: of synthetic medium
- disadvantages:
- uses: important for genetic and specific or
precise studies - disadvantages:
– preparation is time-consuming
– microorganisms grow relatively slow
– prepared only for microorganisms with known
nutritional requirements
- use: complex medium
- advantages:
- use: routine purposes
- advantages:
– easy to prepare
– support rapid growth of most microorganisms
Types of Culture Media
(based on principal purpose, function, or application)
general purpose
differential
selective
enrichment
assay
Can support most
or almost all
types of species
Nutrient Agar,
Tryptic Soy
Agar, Brain
Heart Infusion
Agar
General purpose
Can distinguish
visually one type
of bacterium
from another
Differential
Allows growth of
a specific type of
microorganism only
selective
Used to increase the number
of microorganisms with
unusual physiological
characteristics; contains
special nutrients for
microorganisms of interest
Cellulose agar,
Petroleum broth, Blood
agar
Enrichment
Used for______ of vitamins,
amino acids, antibiotics; may
be used for qualitative or
quantitative production of a
compound by a microorganism
fermentation media,
TSI agar, Vitamin B12
assay medium
Assay
contains selective agents/additives/toxic chemicals
(salts, dyes, antibiotics, and other inhibitors)
– sodium azide, potassium tellurite, thallium acetate (0.1-0.5 g/L), crystal violet (2 mg/L), penicillin (5-50 units/mL)
- extreme pH value or unusual carbon source to favor growth of a particular organism
- i.e. Thayer-Martin agar (Neisseria gonorrhoeae); NA
with penicillin (Gram-negative bacteria); Thiosulfate
selective medium
- identifies microorganisms by the appearance of
their colonies and exploits the ability of a
particular microorganism to change the appearance of the medium - with special reagents like pH indicators and dyes
- i.e. blood agar (Streptococcus species); Mac-
Conkey agar (E. coli and lactose fermenters)
differential medium
contains general nutrients and 5% sheep blood.
It is useful for cultivating fastidious organisms and for determining the hemolytic capabilities of an organism.
Blood agar
Some bacteria produce exoenzymes that lyse red blood cells and degrade hemoglobin;
hemolysins.
breaks down the red blood cells and hemoglobin
completely.
Beta-hemolysin
Beta-hemolysin leaves a clear zone around the bacterial growth. Such results are referred to as
β-hemolysis (beta hemolysis).
partially breaks down the red blood cells and leaves a greenish color behind.
Alpha-hemolysin
The greenish color is caused by the presence of biliverdin, which is a by-product of the breakdown of hemoglobin.
α-hemolysis (alpha hemolysis).
If the organism does not produce hemolysins and does not break down the blood cells, no clearing will occur.
γ-hemolysis (gamma hemolysis).
enrichment contains special nutrient(s) for the microbe of interest
and inhibitory substances to suppress unwanted microorganisms
cellulose, petroleum, blood
- used for the assay of vitamins, amino acids,
antibiotics, etc.; used for qualitative or
quantitative production of such a compound by
a microorganism - of prescribed composition
- i.e. fermentation media, Triple Sugar Iron
Agar, Media for Antibiotic Sensitivity Testing,
Vitamin B12 Assay Medium
Assay Medium
Other Types of
Culture Media
Indicator Medium
Sugar Medium
Transport Medium
Biochemical Reaction Medium
- medium contains an indicator which
changes its color when a bacterium
grows in them - i.e. Blood agar (also a differential
media); Mac Conkey’s medium
Indicator Medium
- medium containing any fermentable substances (i.e.
glucose, arabinose, lactose, starch) - consists of 1% of the sugar in peptone water
- contain a small tube (Durham tube) for the detection
of gas by the bacteria
Sugar Medium
A differential medium that contains 1% lactose,1% sucrose, 0.1% glucose, ferrous sulfate and pH indicator phenol red
performed in gram negative negative bacteria
used to differentiate enteric based on ability to reduce sulfur and ferment carbs.
Triple sugar Iron Test
A differential medium that contains 1% lactose,1% sucrose, 0.1% glucose, ferrous sulfate and pH indicator phenol red
performed in gram negative negative bacteria
used to differentiate enteric based on ability to reduce sulfur and ferment carbs.
Triple sugar Iron Test (TSI)
a.TSI Red/red-
b.control
c. Red/yellow-
d. Yellow/yellow-
e. Red/yellow with H2S
a. no sugar fermentation
c. glucose fermented but lactose and sucrose not fermented
d. glucose fermented. lactose and/or sucrose fermented
medium used for transporting
samples (prevent microbial
proliferation; maintain viability
of microorganisms)
- i.e. Stuart’s medium (non nutrient
soft agar gel containing a reducing
agent); buffered glycerol saline (for
enteric bacilli)
Transport Medium
recommended for the preservation and
transportation of Neisseria species and other fastidious organisms from the
clinic to laboratory.
This medium is a chemically defined, semisolid, non-nutrient medium which
prevent microbial proliferation.
Transport Medium Stuart
The transport medium provides an adequate degree of ______ which can be monitored by means of the redox indicator methylene blue.
anaerobiosis
- medium used to provide additional information for the identification of the bacterium
- i.e. Triple sugar iron agar (sugar fermentation);
SIM Medium (Indole test); Citrate utilization;
Christensens urease medium (Urease test)
Biochemical Reaction Medium
Yellow – Acid
Pink - Alkaline
a. Yellow slant / Yellow butt (A/A) –
b. Pink slant / Yellow butt (K/A) –
c. Pink slant / no colour change (K/K) –
d. Black colour –
( TSI)
e. Gas bubbles or crack in the medium –
f. LF –
g. NLF –
h. H2S -
a. Lactose
fermenters.
b. Non lactose
fermenters
c. non-fermenters
d. H2S production
e. gas production
f. E.coli, Klebsiella
g. Salmonella, Shigella
h. Proteus
- Used to detect indole production by
the organism. - They produce indole from tryptophan
present in peptone water. - After overnight incubation, a few
drops of indole reagent (Kovac’s
reagent) is added. - Positive test is indicated by a pink ring
indole test
- Indole positive –
- Indole negative –
E.coli
Klebsiella, Salmonella
– Positive indole test –
– Negative indole test -
pink ring
yellow ring
SIM medium result:
S
a.black
b.colorless
I
a.Red with kovac’s
b. colorless
M
a. organism growing only in line of inoculation
b. organism appears as haze beyond line of inoculation
S
a.positive for cysteine defulrase production
b. negative or cysteine defulrase production
I
a.positive for tryptophanase production
b.negative for tryptophanase production
M
a. non-motile
b.motile
- Done in Simmon’s Citrate medium.
- To detect the ability of certain
bacteria to utilize citrate as the
sole source of carbon. - Contains Sodium citrate and
bromothymol blue as the indicator. - If citrate is utilized, alkali is
produced which turns the medium to
blue.
– Citrate positive – blue color
– Citrate negative – green color - Positive – Klebsiella
- Negative – E.coli
CITRATE UTILIZATION TEST
- Done in Christensen’s urease
medium. - This test is used to detect
organisms that produce urease. - Urease produced by the
organisms split urea into ammonia
and CO2.
– Urease positive – pink color
– Urease negative – yellow color - Positive – Proteus, Klebsiella
- Negative – E.coli, Salmonella
UREASE TEST
– Urease positive –
– Urease negative –
* Positive –
* Negative –
pink color
yellow color
Proteus, Klebsiella
E.coli, Salmonella
– Citrate positive –
– Citrate negative –
* Positive –
* Negative –
blue color
green color
Klebsiella
E.coli
- These media are used to grow
anaerobic organisms. - e.g: Robertson’s cooked meat
medium, Thioglycolate medium.
Anaerobic media
are anaerobic organism
clostridia
optimum temp. for growth of anaerobic
pH
37’C
7-7.4
most organism produce gas in this medium
saccharolytic species turn meat pink
proteolytic species turn meat black with foul smell
anaerobic media
example of anaerobes attack meat proteolytic
Cl. tetani
example of anaerobes attack carbohydrates in meat saccharolytic
clostridium perfringens
hydrogen ion concentration
* pH values less than 7 – acidic
* pH values greater than 7 - basic
* optimum pH for most bacteria is near
neutrality (pH 7)
* cytoplasm of most bacteria is pH 7
pH
pH range
Acidophiles
Neutrophiles
Alkaliphiles
Acidophiles < pH 5.4
Neutrophiles pH 5.4 - 8.5
Alkaliphiles pH 7.5 – 11.5
– acid-loving organisms
– can be found in acidic lakes, gastrointestinal tract
– most fungi (acid-tolerant; optimum temperature 5 or below)
– some algae, bacteria, and several Archaea
acidophiles
– acid-loving organisms
– can be found in acidic lakes, gastrointestinal tract
– most fungi (acid-tolerant; optimum temperature 5 or below)
– some algae, bacteria, and several Archaea
– high H+ concentration is required to maintain cell membrane
stability
acidophiles
– some algae, bacteria, and several Archaea pH
- Lactobacillus
- Helicobacter pylori
- Acidithiobacillus (sulfur-oxidizing bacteria)
- red alga Cyanidium caldarium, green alga Dunaliella acidophila
- fungi: Aconitum cylatium, Cephalosporium sp., Trichosporon cerebriae
- archaea: Sulfolobus and Thermoplasma, Picrophilus
- Lactobacillus (pH 6)
- Helicobacter pylori (pH 2 or less)
- Acidithiobacillus (sulfur-oxidizing bacteria) (pH <4)
- red alga Cyanidium caldarium, green alga Dunaliella acidophila (pH <1)
- fungi: Aconitum cylatium, Cephalosporium sp., Trichosporon cerebriae
(near pH 0) - archaea: Sulfolobus and Thermoplasma, Picrophilus (negative pH values)
Acidophile examples
-Lactobacillus
-fungi
-Helicobacter pylori
-Acidithiobacillus thiooxidans
-Thermoplasma
-Picrophilus
-Sulfolobus acidocaldarius
most human disease-causing bacteria
(human blood and tissues pH = 7.2 – 7.4)
protozoans and most bacteria (pH 6.5-7.5)
neutrophiles
examples of neutrophiles
-E. coli bacteria in gut
-Balantidium coli (protozoan) in
human large intestines
-Salmonella bacteria on tissue surface
-Staphylococcus skin infection
base-loving organisms
live in soda lakes, high-carbonate soils
i.e. Bacillus, Vibrio cholerae (pH 9), Alcaligenes
faecalis (>pH 9), Agrobacterium (pH 12)
some produce hydrolytic enzymes (proteases and lipases)
alkaliphiles
examples of alkaliphiles
-Vibrio cholerae
-Agrobacterium
-Alcaligenes faecalis
- one of the most, if not the most, important
environmental factors affecting growth and
survival of microorganisms
Temperature
three critical temperatures (affecting enzyme
function) or cardinal temperatures:
-minimum growth temperature
-optimum growth temperature
-maximum growth temperature
lowest temperature
at which cells can divide (a)
* membranes solidify; slow transport process thus
growth could not occur
minimum growth temperature
– temperature at
which cells divide most rapidly (b)
* enzymatic reaction occurring at maximal possible
rate
optimum growth temperature
– highest temperature
at which cells can divide (c)
* protein denaturation, collapse of cell membrane,
cell lysis
maximum growth temperature
Temperature Classes of
Microorganisms
Psychrophiles:
Mesophiles
Thermophiles
Hyperthermophiles
Psychrophiles: <0 to 20’C 15’C
Mesophiles 10 to 48’C 37’C
Thermophiles 40 to 72’C 60’C
Hyperthermophiles 65 to 110’C 80’C
“cold-loving organisms”
grow best at -10 ̊ to 20 ̊C
live mostly in cold water and soil (Arctic and
Antarctic regions) and can cause spoilage of
refrigerated food
psychrophiles
