GROWTH IN VITRO
Factors that affect Microbial Growth
FACTORS THAT AFFECT MICROBIAL
availability of nutrients- living organisms require
nutrients. Appropriate nutrients must be
available to survive in particular environment
Moisture- all living organisms require water to
carry out their normal metabolic processes and
most will die in environments containing too
– every microorganisms growth
thermophiles- organisms that love heat. (minimum growth
temperature: 25oC, optimum growth temperature: 5060oC, maximum growth temperature: 113oC)
mesophiles- microbes that grow best at moderate
temperatures (minimum growth temperature:
10oC, optimum growth temperature: 20-40oC, maximum
growth temperature: 45oC)
psychrophiles- prefer cold temperatures. They thrive in
cold ocean water.(minimum growth temperature:5oC, optimum growth temperature: 10-20oC, maximum
growth temperature: 30oC)
psychroduric- microbes that prefer warmer
temperatures, but can tolerate or endure very cold
temperatures and can be preserved in the frozen state.
refers to the acidity and alkalinity of
a solution. Most microbes prefer a neutral
or slightly alkaline growth medium (pH
7.0 to 7.4)
acidophiles- such as those that can live in highly
acidic environments such as pH 2 to 5)
alkaliphiles- prefer an alkaline environment 9pH
greater than 8.5), such as is found inside the
atmosphere- to grow a particular
microorganism in the laboratory, it is
necessary to provide the atmosphere that
ENCOURAGING THE GROWTH OF
MICROORGANISMS IN VITRO (EVENTS OUTSIDE
Culture- the growth of organisms obtained in a
culture medium after its incubation period
Culture medium- any material where
microorganisms may thrive for their nourishment
Incubation period- is the time needed to let
previously inoculated culture media to show a
distinct colony or colonies within a desired
temperature. It is also the time needed for the
microorganisms to adapt, grow and multiply in a
Colony- a group of microorganisms growing
together characteristically in a culture medium
Inoculum- the fished colony hanging on a wire
loop, cotton swabs that is ready for transfer to
another culture medium for cultivation.
Types of culture:
Contaminated culture- a culture that accidentally
contains one or more group of microorganisms
which should not be there at all.
Mixed culture- there are two or more desired
species of microorganism living in the culture
Pure culture- a culture that contains only one
group of microorganisms.
CULTURING BACTERIA IN THE LABORATORY
Bacterial growth refers to an increase in the number of
organisms rather than an increase in their size. When each
bacterial cell reaches its optimum size, it divides by binary
fission into two daughter cells. On solid medium, binary
fission continues through many generations until a colony
is produced. A bacterial colony is a mound or pile of
bacteria containing millions of cells. Binary fission
continues for as long as the nutrient supply, water, and
space allow and ends when the nutrients are depleted or
the concentration of cellular waste products reaches a toxic
level. Colony development on agar surfaces helps in the
identification of the cultured microorganism.
Typically, depending on the medium used, individual
species of microorganisms form colonies of characteristic
size and shape. Even when mixed population has been
properly cultured, one can distinguish one from the other
based on overall appearance of the colonies.
The media that are used in microbiology laboratories
to culture bacteria are referred to as artificial media
or synthetic media, because hey do not occur
naturally; rather, they are prepared in the laboratory.
They are number of ways of categorizing the media that
are used to culture bacteria:
chemically defined medium- is one in which all the
ingredients are known; this is because the medium was
prepared in the laboratory by adding a certain number of
grams of each of the components(carbohydrates, amino
complex medium- is one in which the exact contents are not
known. Complex media contain ground up or digested extracts
from animal organs (hearts, liver, and brains), fish, yeasts, and
plants, which provide the necessary nutrients, vitamins and
Liquid media- also known as broths are contained in tubes
Ex.: thioglycollate broth is very popular liquid medium for
use in the laboratory. THIO supports the growth of all
categories of bacteria from obligate aerobes to obligate
Solid media- are prepared by adding agar to liquid media
and then pouring the media into tubes or Petri
dishes, where the media solidifies. Bacteria are then
grown on the surface of the agar- containing solid media. A
gar is a complex polysaccharide that is obtained from a red
marine alga; it is used as a solidifying agent, much like
gelatin is used as a solidifying agent in the kitchen.
Enriched medium is a broth or slid medium containing a
rich supply of special nutrients that promotes the growth of
fastidious organisms (complex nutritional requirements) It
is usually prepared by adding extra nutrients to a medium
called nutrient agar.
Ex. Blood agar (nutrient agar plus 5% sheep red blood
cells) and chocolate agar (nutrient agar plus powdered
hemoglobin) Chocolate agar is used to culture
important, fastidious, bacterial pathogens, like Neisseriae
gonorrhoeae and Haemophilus influenzae.
selective medium has added inhibitors that
discourage the growth of certain organisms without
inhibiting growth of an organism being sought.
Ex. MacConkey agar inhibits the growth of gram
positive bacteria and thus is selective for gramnegative bacteria.
Phenylethyl alcohol (PEA agar and colistin-nalidixic
acid (CAN) agar inhibit the growth of Gram-negative
bacteria and thus are selective for gram-positive
Thayer Martin agar and Martin Lewis agar (chocolate
agars containing extra nutrients plus several
antimicrobial agents) are selective for Neisseria
gonorrhoeae. Only salt –tolerant (haloduric) bacteria
can grow on mannitol salt agar (MSA)
Differential medium- permits the differentiation of
organisms that grow on the medium.
EX. MacConkey agar is frequently used to differentiate among
various gram-negative bacilli that are isolated from fecal
Gram-negative bacteria able to ferment lactose (an ingredient
of MacConkey agar) produce pink colonies, whereas those
unable to ferment lactose produce colorless colonies.
It differentiates between lactose-fermenting and non-lactose
fermenting gram negative bacteria.
Mannitol salt agar is used to screen for Staphylococcus
aureus; not only will S. aureus grow on MSA, but it turns the
originally pink medium to yellow because of its ability to
Blood agar is also a differential medium because it is used to
determine the type of hemolysis that bacterial isolate
Inoculation of Culture Media
Inoculation of a liquid medium involves adding a
portion of the specimen to the medium. Inoculation of
a solid or plated medium involves the use of a sterile
inoculating loop to apply a portion of the specimen to
the surface of the medium, process called streaking.
Importance of using Sterile Technique:
sterile technique is practiced when it is necessary to
exclude all microorganisms from a particular area, so that
the area will be sterile. Such unwanted microbes are
referred to as contaminants.
Minimizes the possibility of contamination and protects the
laboratory worker from becoming infected.
After media are inoculated, they must be incubated.
Incubator contains the appropriate atmosphere and
moisture level and is set to maintain the appropriate
temperature. To culture most human pathogens, the
incubation is set at 35 to 37oC.
3 TYPES OF INCUBATOR
A carbon dioxide incubator – concentration of 5 to
10% carbon dioxide. To isolate capnophiles.
A non CO2 incubator is an incubator containing room
air; thus it contains atmosphere devoid of oxygen.
an anaerobic incubator is an incubator containing an
atmosphere devoid of oxygen.
Bacterial Population growth curve
A population growth curve for any particular species of bacteria may
be determined by growing a pure culture of the organism in a liquid
medium at a constant temperature.
The growth curve four phases:
Lag phase- bacteria absorb nutrients, synthesize enzymes and
prepare for cell division. The bacteria do not increase in number
during the lag phase
Logarithmic growth phase- the bacteria multiply rapidly that
the umber of organisms doubles with each generation time.
Growth rate is the greatest during the log phase. The log phase is
always brief, unless the rapidly dividing culture is maintained
by constant addition of nutrients and frequent removal of waste
stationary phase -as the nutrients in the liquid medium are
used up and the concentration of toxic waste products from the
metabolizing bacteria build up, the rate of division slows, such
that the number of bacteria that are dividing equals the number
that are dying.
Death phase - as overcrowding occurs the concentration of toxic
waste products continues to increase and the nutrient supply
decreases. The microorganisms die at a rapid rate.