The document discusses factors affecting microbial growth in vitro, including nutrient availability, moisture, temperature, pH, and atmospheric conditions. It describes various types of culture media used in microbiology, such as enriched, selective, and differential medias, and outlines the process of culturing bacteria, including inoculation and incubation. Additionally, it explains the bacterial population growth curve, which consists of four phases: lag, logarithmic growth, stationary, and death phases.

1. CONTROLLING MICROBIAL GROWTH
IN VITRO
Factors that affect Microbial Growth

2. FACTORS THAT AFFECT MICROBIAL GROWTH
 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 little moisture.

3.  Temperature – every microorganisms growth
temperature
 thermophiles- organisms that love heat. (minimum growth
temperature: 25oC, optimum growth temperature: 50-60oC,
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.

4.  pH- 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 intestine(pH 9)
 gaseous atmosphere- to grow a particular
microorganism in the laboratory, it is
necessary to provide the atmosphere that it
requires.

5. ENCOURAGING THE GROWTH OF
MICROORGANISMS IN VITRO (EVENTS OUTSIDE
THE BODY)
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 and reproduction
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 new environment.
Colony- a group of microorganisms growing together
characteristically in a culture medium

6.  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:
1. Contaminated culture- a culture that accidentally
contains one or more group of microorganisms which
should not be there at all.
2. Mixed culture- there are two or more desired species
of microorganism living in the culture medium
3. Pure culture- a culture that contains only one group of
microorganisms.

7. 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.

8. Culture Media
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 acids, salts)
 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
minerals.
 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 anaerobes.

9.  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.

10.  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 gram-negative
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 bacteria
 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)

11.  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
specimens.
 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 ferment mannitol.
 Blood agar is also a differential medium because it is used to
determine the type of hemolysis that bacterial isolate produces.

12. 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.
Incubation
 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.

13.  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.

14. 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 products
 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.