Controlling

Microbial Growth
in Vitro
 FACTORS THAT AFFECT MICROBIAL
GROWTH
• Availability of nutrients
• Moisture
• Temperature; thermophiles (organisms that love heat);
hyperthermophiles (Pyrolobus fumarii); mesophiles;
psychrophiles; psychroduric organisms
Categories of Bacteria on the Basis of Growth Temperature
Category Minimum Growth Optimum Growth Maximum Growth
Temperature Temperature Temperature
Thermophiles 25°C 50° - 60°C 113°C
Mesophiles 10°C 20° - 40°C 45°C
Psychrophiles -5°C 10° - 20°C 30°C
• pH; acidophiles (2 – 5); alkaliphiles (>8.5) (Vibrio
cholera)
• Osmotic pressure and Salinity (plasmolysis,
plasmoptysis); halophilic; haloduric
• Barometric pressure ; piezophiles
• Gaseous atmosphere ; microaerophiles;
capnophiles
 Encouraging the Growth of Microbes In
• Vitro
Bacterial Growth
- Proliferation or Multiplication of bacteria
- E.coli, V. cholera, Staphylococcus spp., and Streptococcus spp
(GT- 20mins)
- Pseudomonas and Mycobacterium spp (GT – 10mins)
- Mycobacterium tuberculosis (GT – 18-24 hours)
 microorganisms that are difficult to grow in the laboratory
are said to be fastidious
• Culture Media
- Are used in the laboratory to culture bacteria
- artificial or synthetic
 classification:
 chemically defined medium
 complex medium
 enriched medium: blood agar and chocolate agar
 selective medium: MacConkey agar, PEA agar and CNA agar, Thayer-
Martin agar and Martin-Lewis agar (N. gonorrhoeae), MSA
(haloduric)
 differential medium
Agar – is a complex polysaccharide that is obtained from a red marine
alga
 categories:
 liquid media (broths)
 solid
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 a specimen to the
surface of the medium—streaking
Importance of Using “Aseptic Technique”
Prevent:
a. microbiology professionals from becoming infected
b. contamination of their work environment
c. contamination of clinical specimens, cultures, and
subcultures
Incubation
types:
 carbon dioxide incubators
 non-carbon dioxide incubators
 anaerobic incubators
 Bacterial Population Counts
• Spectrophotometer
• viable plate count – is used to determine the total number
of viable bacteria in a liquid sample
Bacterial Population Growth Curve
4 phases:
I. lag phase – bacteria absorb nutrients, synthesize enzymes, and
prepare for cell division
II. logarithmic growth phase – bacteria multiply rapidly
III. stationary phase – the culture is at its growing density
IV. death phase – culture may die completely or some may
survive for months
Culturing Viruses and Other Obligate
Intracellular Pathogens in the Lab
Obligate intracellular pathogens
- are microbes that can survive and multiply only within
living cells (host cells)
- include viruses and two groups of Gram-negative
bacteria—rickettsias and chlamydias
• culturing these organisms in the laboratory is a
challenge: they must be grown in embryonated
chicken eggs, laboratory animals, or cell cultures
 Culturing Fungi in the Laboratory
• Fungi (including yeasts, moulds, and dimorphic fungi) grow on
and in a variety of solid and liquid culture media
• There is no single medium that is best for all medically important
fungi
• Examples of culture media for fungi includes: brain heart infusion
(BHI) agar, BHI with blood, and Sabouraud dextrose agar (SDA);
due to its low pH, SDA is selective for fungi
• caution must be exercised when culturing fungi – some are highly
infectious
Culturing Protozoa in the Laboratory

Most microbiology laboratories do not culture protozoa;

some research and reference labs do, however.
• Examples of protozoa that can be cultured in vitro are
amebae, Giardia lamblia, Leishmania spp., Toxoplasma
gondii, Trichomonas vaginalis, and Trypanosoma cruzi.
• Due to the severity of diseases that they cause, it is of
greatest importance to culture amebae: Acanthamoeba
spp., Balamuthia spp., and Naegleria fowleri
 Inhibiting the Growth of Microbes In Vitro
• Sterilization – involves the destruction or elimination of all
microbes
• Disinfection – describes the elimination of most or all pathogens
from nonliving objects
• Disinfectants – chemicals used to disinfect inanimate objects
• Antiseptics – are solutions used to disinfect skin and other living
tissues
• Sanitization – is the reduction of microbial populations to levels
considered safe
Microbicidal Agents
• Bactericidal agents – specifically kill bacteria
• Sporicidal agents – kill bacterial endospores
• Fungicidal agents – kill fungi, including fungal spores
• Algicidal agents – used to kill algae in swimming polls
and hot tubs
• Viricidal agents – destroy viruses
• Pseudomonicidal agents – kill Pseudomonas spp.
• Tuberculocidal agents – kill M. tuberculosis
Microbistatic Agents
- is a drug or chemical that inhibits reproduction of
microorganisms, but does not necessarily kill them

• Bacteriostatic agent – is one that specifically inhibits the

metabolism and reproduction of bacteria
• Lyophilization – is a process that combines dehydration
and freezing; a good method of preserving
microorganisms for future use
Definition of terms:
• Sepsis – refers to the presence of pathogens in blood or tissues
• Asepsis – means the absence of pathogens
• Aseptic techniques – are used to eliminate and exclude
pathogens
• Antisepsis – is the prevention of infection
• Antiseptic techniques – developed by Joseph Lister in 1867,
refers to the use of antiseptics
• Sterile technique – is practiced when it is necessary to exclude
all microorganisms from a particular area, so that the area will
be sterile
Physical Methods to Inhibit Microbial
Growth
Heat – most common type of sterilization for inanimate
objects able to withstand high temperatures
2 factors: Temperature & Time

 Thermal Death Point (TDP) – is the lowest temperature

that will kill all the organism in a standardized pure culture
within a specified period
 Thermal Death Time (TDT) – is the length of time
necessary to sterilize a pure culture at a specified
temperature
• Dry Heat – dry heat baking in a thermostatically controlled
oven provides effective sterilization of metals, glasswares,
some powders, oils and waxes
• Moist Heat – heat applied in the presence of moisture, as
in boiling or steaming, is faster and more effective than dry
heat and can be accomplished at a lower temperature; thus
it is less destructive to many materials that would damage
at higher temperatures
• Cold – refrigeration merely slows the growth of most
microorganisms; it does completely inhibit the growth
• Desiccation – the process of being thoroughly dried
• Radiation – the UV rays, which do not penetrate glass and building
materials, are effective only on surfaces. They penetrate cells and
thus can damage DNA
• Ultrasonic waves – frequently used means of cleaning delicate
equipment
• Filtration – filters of various pore sizes are used to filter or
separate cells, large viruses, bacteria and certain other
microorganisms from the liquids or gases in which they are
suspended
• Gaseous atmosphere – it is possible to inhibit growth of
microorganisms by altering the atmosphere in which they are
located
Various Factors Affect the Efficiency or Effectiveness of a
Disinfectant
• Prior cleaning of the object/surface to be infected
• The organic that is present, meaning the presence of organic
matter on the materials being treated
• The bio-burden, meaning the type and level of microbial
contamination
• The concentration of the disinfectant
• The contact time, meaning the amount of time that the
disinfectant must remain in contact with the organisms in order
to kill them. The physical nature of object being disinfected
• Temperature and pH
Characteristics of an Ideal Chemical Anti-Microbial
Agent
• It should kill a variety of microorganisms
• It should be fast acting, contact time should be short
• Should not be affected by the presence of organic material
• Must be nontoxic to human tissues and noncorrosive and non-
destructive to materials
• It must be soluble to water and easy to apply
• Should be inexpensive and easy to prepare
• Should be odourless
• It must be stable both as a concentrate and as a working
dilution