CONTROLLING THE
GROWTH OF MICROBES
CHAPTER 8
FACTORS THAT AFFECT MICROBIAL GROWTH
• Availability of nutrients
• moisture
• desiccation – bacterial endospores and protozoan cysts
• temperature
• pH
• osmotic pressure
• barometric pressure
• composition of the atmosphere
TEMPERATURE
• Thermophiles – microorganisms that grow best
at high temperatures (organisms that love heat)
• Hyperthermophiles
• AKA: Extreme thermophiles
• organisms that favor temperature above 1000C
• Pyrolobus fumarii – an archaeon that lives around
1130C
• Mesophiles – microbes that grow best at
moderate temperatures
TEMPERATURE
• Psychrophiles – are organisms that love cold
temperatures
• e.g. algae (often pink) seen living on snow
• Psychrotrophs – one of the group of
psychrophile that has an optimum growth
temperature of 40C (refrigerator temperature)
• Psychroduric – organisms that prefer warm
temperatures, but can tolerate or endure very
cold temperatures and can preserved in the
frozen state
PH
• pH – refers to the hydrogen ion
concentration of a solution and thus the
acidity or alkalinity of the solution
• Acidophiles –prefer acidic environments
• pH of 2 to 5
• e.g. fungi
• Alkaliphiles – prefer alkaline environments
• pH > 8.5
• Vibrio cholera – the only human pathogen
that grows well above pH 8
OSMOTIC PRESSURE AND SALINITY
• Osmotic pressure – it is the pressure exerted on • Halophilic organisms
a cell membrane by solutions both inside and • “halo” means salt and “philic” means love
outside the cell • microorganisms that prefer salty environment
• Solutes – are substances dissolves in liquids • e.g.Vibrio cholera
• Osmosis – is defined as the movement of a solvent • Haloduric organisms
(e.g. water), through a permeable membrane, from a • organisms that do not prefer to live in salty
solution having a lower concentration of solute to a environments but are capable of surviving there
solution having a higher concentration of solute also
• e.g. Staphylococcus aureus
HYPERTONIC SOLUTION
• it is when the concentration of solutes in the
environment outside a cell is greater than the
concentration of solutes inside the cell; in which the
cell is suspended in the solution
• Crenation – the loss of water causes the cell
(human cell) to shrink, the cell is to be crenated
• Plasmolysis – this condition wherein the bacterial
cell wherein the cell membrane and cytoplasm
shrink away from the cell wall
• it inhibits the bacterial cell growth & multiplication
HYPOTONIC SOLUTION
• it is when the concentration of solutes outside a
cell is less than the concentration of solutes inside
the cell; the solution in which the cell is suspended
• Hemolysis – if sufficient water enters the human
cell such as erythrocytes, it causes the cell to swell
and eventually will burst (lyse)
• Plasmoptysis – wherein the pressure becomes so
great that the bacterial cell ruptures, and some
organelles like cytoplasm escape from the cell
ISOTONIC SOLUTION
• it is when the concentration of solutes
outside a cell is equals the concentration
of solute inside the cell
BAROMETRIC PRESSURE
• Piezophiles
• AKA: Barophiles
• organisms that live in the environments where there
is high barometric/atmospheric pressure, such as at
the bottom of the ocean
• e.g. Archaea
ENCOURAGING THE GROWTH OF MICROBES IN VITRO
CULTURING BACTERIA IN THE LABORATORY
• Ferdinand Cohn, Joseph Schroeter and Oscar • Richard Julius Petri
Brefeld – those who successfully attempts to • invented the glass petri dish that is used
culture microorganisms in a laboratory setting as containers for solid culture media and
bacterial cultures
• Robert Koch – he developed both liquid and solid
form of artificial media • Joseph Lister
• Gelatin – was initially used as solidifying agent in • 1st person to obtain a pure culture of a
Kochs culture media bacterium Streptococcus lactis in a
liquid medium
• Fanny Hesse – commonly called Frau Hesse
• suggested the use of agar
BACTERIAL GROWTH
• refers to an increase in the number of
organisms; multiplication of bacteria
• Bacterial colony – is a mound or pile
of bacteria containing million of cells
GENERATION TIME
• the time taken for one cell to become two cells by • rapid growers – bacteria with short generation
binary fission times
• Escherichia coli,V. cholera, Staphylococcus • slow growers – bacteria with long generation
spp., and Streptococcus spp – have generation time
time of about 20 mins
• fastidious – microorganisms that are difficult to
• Pseudomonas & Clostridium spp. – every 10 grow in the laboratory
minutes • they have complex nutritional requirements
• Mycobacterium tuberculosis – every 18 to 24 • obligate intracellular pathogens
hours • must be inoculated into live animals, embryonated
chicken eggs or cell cultures
CULTURE MEDIA
• Artificial media • Complex medium
• AKA: Synthetic media • in which the exact content are not known; it
• the media that are used in microbiology lab to culture contain ground-up or digested extracts from
bacteria; they don’t occur naturally and are prepared animal organs (e.g. hearts, livers and brains),
in the lab fish, yeasts and plants, which provide the
necessary nutrients, vitamins and minerals
• Chemically defined medium
• in which all the ingredients are known; the medium
was prepared in the lab by adding a certain number of
grams of each of the components (e.g. carbohydrate,
amino acids & salts)
LIQUID & SOLID MEDIA
• Liquid media
• AKA: Broths or Tubed media
• they are contained in tubes
• Solid media
• are prepared by adding agar to liquid media and
then pouring the media into tube or Petri dishes,
where the media solidifies
• Agar – is a complex polysaccharide that is
obtained from a red marine alga, that is used as
solidifying agent
ENRICHED MEDIUM
• is a broth or solid medium containing a rich supply of
special nutrients that promotes the growth of fastidious
organisms
• nutrient agar – usually prepared by adding extra nutrients
to a medium
• Blood agar – nutrient agar plus 5% sheep red blood cells;
bright red color
• Chocolate agar – nutrient agar plus powdered
hemoglobin; brown color (the color of chocolate)
• is used to culture important, fastidious, bacterial pathogens
such as N. gonorrhoeae & H. influenza, which will not
grow on blood agar
SELECTIVE MEDIUM
• there’s added inhibitors that discourage the growth • Thayer-Martin agar & Martin-Lewis agar
of certain organisms without inhibiting the growth • chocolate agars containing extra nutrients plus
of the organisms being sought several antimicrobial agents
• MacConkey agar – it inhibits the growth of • selective for N. gonorrhoeae
Gram-positive bacteria and is selective for Gram- • Mannitol salt agar (MSA)
negative bacteria • only salt-tolerant (haloduric) bacteria that can
• Phenylethyl alcohol (PEA) agar & Colistin- grow on this media
Nalidixic acid (CAN) agar
• it inhibit the growth of Gram-neg bacteria & are
selective for G+ bacteria
DIFFERENTIAL MEDIUM
• it allows one to readily differentiate among the
various types of organisms that are growing on
medium
• MacConkey agar
• differentiates between lactose-fermenting and
non-lactose fermenting G-bacteria
DIFFERENTIAL MEDIUM
• Blood agar
• is also a differential medium because it is
used to determine the type of
hemolysis (alteration or destruction of
red blood cells) that the bacterial isolate
produces
THIOGLYCOLLATE BROTH
• is a very popular liquid medium for use
in the bacteriology laboratory
• it supports the growth of all categories
of bacteria from obligate aerobes to
obligate anaerobes
INOCULATION OF CULTURE MEDIA
• Inoculation of liquid medium
• it involves adding a portion of the specimen to
the medium
• Inoculation of solid or plated medium
• it involves the use of sterile inoculating loop to
apply a portion of the specimen to the surface of
the medium; a process known as streaking
IMPORTANCE OF USING ASEPTIC TECHNIQUE
• Aseptic technique
• is practiced in the microbiology laboratory to prevent
infection of individuals and contamination of the work
environment, clinical specimens and cultures
• contaminants – refer to unwanted organisms; and
the plate is said to be contaminated
• Biologic safety cabinet (BSC)
• inoculating media using this tool minimizes the
possibility of contamination and protects the
laboratory worker from becoming infected with the
organisms that he/she working with
INCUBATION
• after media are inoculated, they must be incubated (i.e.
they must be placed into a chamber (called
incubator) that contains the appropriate
atmosphere and moisture level and is set to maintain
the appropriate temperature
• three types of incubators:
• CO2 incubators
• non-CO2 incubators
• anaerobic incubators
CO2 INCUBATOR
• is an incubator with a CO2 cylinder
attached
• this incubator is used to isolate
capnophiles
NON-CO2 INCUBATOR
• is an incubator containing room air
• contains about 20% to 21% oxygen
ANAEROBIC INCUBATOR
• is an incubator containing an atmosphere
devoid of oxygen
• Pure culture – is a culture that
contains only one species of organisms
BACTERIAL POPULATION COUNTS
• the microbiologist may determine:
• the total number of bacterial cells in the liquid (the total
would include both viable and dead cells)
• the number of viable (living) cells
• Spectrophotometer – wherein a beam of light is
passed through the liquid
• Turbidity increases as the number of organisms increases;
therefore, the amount of transmitted light decreases as the
bacteria increase in number
• which is usually expressed as the number of organisms per
milliliters of suspension
VIABLE PLATE COUNT
• is used to determine the number of viable
bacteria in a liquid sample, such as milk, water,
ground food diluted in water, or a broth culture
• viable cell count
• is an important part of a urine culture
• the number of viable bacteria per milliliter of a
urine specimen is used as an indicator of a urinary
tract infection
BACTERIAL POPULATION GROWTH CURVE
• consists of 4 phases:
• LAG phase
• the 1st phase of the growth curve wherein the
bacteria absorb nutrients, synthesize enzymes and
prepare for cell division
• the bacteria do not increase in number
• LOG phase
• AKA: Logarithmic growth phase/exponential growth
phase
• the bacteria multiply so rapidly that the number of
organisms doubles with each generation time
BACTERIAL POPULATION GROWTH CURVE
• Stationary phase
• it is during this phase that the culture is at its greatest
population density
• Death phase
• AKA: Decline phase
• in this phase wherein the microorganisms die at rapid rate
• L-phase variants (L-forms)/protoplasts/spheroplasts
• wherein some cells undergo involution and some various
shapes, becoming long, filamentous rods or branching or
globular forms that are difficult to identify, some develop w/o
a cell wall
CHEMOSTAT
• used for continuous cultures in a controlled
environment, which regulates the supply of nutrients and
the removal of waste products and excess
microorganisms
• are used in industries where yeast is grown to produce
beer and wine, where fungi and bacteria are cultivated to
produce antibiotics, where E.coli cells are grown for
genetic research and in any other process needing a
constant source of microorganisms
CULTURING VIRUSES IN LABORATORY
• cell line
• is a permanently established cell culture that will
proliferate indefinitely given appropriate fresh medium
and space
• ex: kidney cells from monkeys, rabbits, or humans;
human and mink lung cells and various cancer cell lines
• cytopathic effect (CPE)
• a given virus will cause specific morphologic alterations
to the cells
• e.g. rounding, swelling and shrinking of cells or cells
may become granular, glassy, vacuolated or fused
CULTURING OBLIGATE INTRACELLULAR PATHOGENS IN LAB
• Can be propagated in the laboratory
using embryonated chicken eggs,
laboratory animals or cell cultures
CULTURING FUNGI IN THE LABORATORY
• Fungi- including yeasts, moulds and dimorphic
fungi will grow on and in various solid and liquid
culture media
• Brain-heart infusion (BHI) agar with blood and
Sabouraud dextrose agar – examples of solid
culture media used to grow fungi
• pH 5.6 – the low pH of SDA inhibits the growth of
most bacteria
CULTURING PROTOZOA IN THE LABORATORY
• Amebae (e.g. Acanthamoeba spp., Balamuthia spp.,
Entamoeba histolytica and Naegleria fowleri), Giardia
lamblia, Leishmania spp.,Toxoplasma gondii,
Trichomonas vaginalis and Trypanosoma cruzi –
examples of protozoa that can be cultured in vitro
• Acanthamoeba, Balamuthia and N. fowleri
• is of greatest importance to culture these following
protozoa in a clinical microbiology laboratory
• these amebae can cause serious (often fatal) infections of
the CNS – infections that are difficult to diagnose by other
methods
INHIBITING THE GROWTH OF MICROBES IN VITRO
DEFINITION OF TERMS
• Sterilization – involves destruction or elimination
of all microbes, including cells, spores and viruses
• Principal sterilizing agents in health care
facilities:
• Dry heat, autoclaving (steam under pressure), ethylene
oxide gas and various liquid chemicals (such as
formaldehyde)
• Types of radiation – ultraviolet (UV) light and
gamma rays
DISINFECTION, PASTEURIZATION, DISINFECTANTS,
ANTISEPTICS AND SANITIZATION
• Disinfection – it involves the elimination of most or • Antiseptics – are solutions used to disinfect skin
all pathogens (except bacterial spores) from and other living tissues
nonliving objects • Sanitization – is the reduction of microbial
• Pasteurization – the method of disinfecting liquids populations to levels considered safe by public health
• the heating process developed by Pasteur to kill standards, such as those applied to restaurants
microbes in wine
• Disinfectants – chemicals used to disinfect
inanimate objects such as bedside equipment and
operating rooms
• do not kill spores (i.e., they are not sporicidal)
MICROBICIDAL AGENTS
• agents having the suffix “-cidal”, “-cide” refers to • Fungicidal agents (fungicides)
killing • kill fungi, including fungal spores
• General terms like: Germicidal agents (germicides), • Algicidal agents (algicides)
biocidal agents (biocides) • used to kill algae in swimming pools and hot tubs
• Microbicidal agents (microbicides) • Viricidal agents (virucidal agents)
• are disinfectants or antiseptics that kill microbes • destroy viruses
• Bactericidal agents (bactericides) • Pseudomonacidal agents – kill Pseudomonas
• specifically kill bacteria, but not the bacterial species
endospores
• Tuberculocidal agents – kill M. tiberculosis
• Sporicidal agents – are required to kill bacterial
endospores
MICROBISTATIC AGENTS
• suffix –”static” inhibit growth and reproduction • Lyophilization – is a process that
• is a drug or chemical that inhibits reproduction of combines dehydration (drying) and
microorganisms, but does not necessarily kill them freezing
• is widely used in industry to preserve
• it inhibits the metabolism and reproduction of foods, antibiotics, antisera,
bacteria microorganisms, and other biologic
• Freeze drying (lyophilization) and rapid materials
freezing (using liquid nitrogen) – are microbistatic
techniques that are used to preserve microbes for
future use or study
SEPSIS, ASEPSIS, ASEPTIC TECHNIQUE, ANTISEPSIS
AND ANTISEPTIC TECHNIQUE
• Sepsis – refers to the presence of pathogens in • Antiseptic technique
blood or tissues • is a type of aseptic technique
• Asepsis – refers to the absence of pathogens • Lister used dilute carbolic acid (phenol) to
cleanse surgical wounds and equipment and a
• Aseptic techniques – are used to eliminate and carbolic acid aerosol to prevent harmful
exclude pathogens microorganisms from entering the surgical field or
• Antisepsis – is the prevention of infection contaminating the patient
• Antiseptic technique – refers to the use of • Sterile technique
antiseptics • is practiced when it is necessary to exclude all
microorganisms from a particular area, so that the
• developed by Joseph Lister
area will be sterile
USING PHYSICAL METHODS TO INHIBIT
MICROBIAL GROWTH
• Physical Methods
• commonly used in hospitals, clinics and
laboratories to destroy or control pathogens that
include:
• Heat
• combination of heat and pressure
• desiccation
• radiation
• sonic disruption
• filtration
HEAT
• it is the most common type of sterilization for
inanimate objects that can withstand high
temperatures
• two factors: Temperature & time
• Thermal death point
• it is the lowest temperature that will kill all the
organisms in a standardized pure culture within a
specified period
• Thermal death time – is the length of time
necessary to sterilize a pure culture at a specified
temperature
DRY HEAT
• a thermostatically controlled oven provides effective
sterilization of metals, glassware, some powders, oils and
waxes
• these items must be bake at 1600C to 1650C for 2 hours
or at 1700C to 1800C for 1 hour
• Incineration (burning) – is an effective means of
destroying contaminated disposable materials
• Flaming the surface of metal forceps and wire
bacteriologic loops
• Open flames
MOIST HEAT
• heat applied in the presence of moisture, as
in boiling or streaming
• it is more effective than dry heat and can be
accomplished at a lower temperature
• it causes proteins to coagulate
• the vegetative forms of most pathogens are
destroyed by boiling for 30 mins
AUTOCLAVE
• is like a large metal pressure cooker that uses steam
under pressure to completely destroy all microbial
life
• it should be set to run 20 minutes at a pressure of
15 psi and a temperature of 121.50C
• Pressure-sensitive autoclave tape &
commercially available strips or solutions
containing bacterial spores – can be used as quality
control measures to ensure that autoclaves are
functioning properly
AUTOCLAVE
BIOLOGIC INDICATOR USED TO MONITOR THE
AUTOCLAVE TAPE
EFFECTIVENESS OF AUTOCLAVING
AUTOCLAVE
BOTULISM FOOD POISONING HOT WATER (>600C)
• is preventable by properly washing and • an effective way to disinfect clothing, bedding
pressure cooking (autoclaving) food and dishes with detergent or soap and to
agitate the solution around the items
• results from the ingestion of Clostridium
botulinum toxins
• The best way to remove microbes from a
kitchen sponge is to rinse it, wring it out, and
microwave it for 30 to 60 seconds
COLD
• Refrigeration cannot be relied upon to kill microorganisms
• it slows their metabolism and their rate of growth
• slow freezing - -240C or above
• causes ice crystals to form within cells and may rupture the
cell membranes and cell walls of some bacteria
• should not be used as a way to preserve or store bacteria
• Rapid freezing
• -250C or less
• using liquid nitrogen, is a good way to preserve foods,
biologic specimens and bacterial cultures
DESICCATION
• In a hospital setting, dried clinical specimens
and dust may contain viable microorganisms
• dry dusting
• important precautions must be observed
include wet mopping of floors, damp dusting of
furniture, rolling bed linens and towels carefully,
and proper disposal of wound dressings
RADIATION
• UV rays – it penetrate cells and can cause damage to
DNA
• genes may be so severely damaged that the cell dies
(especially unicellular microorganisms) or is drastically
changed
• do not penetrate cloth, metals and glass, these metals may
be used to protect persons working in a UV environment
• UV lamp – AKA: Germicidal lamp
• is useful for reducing the number of microorganisms in the
air
• can cause serious burns and cellular damage; skin cancer
RADIATION
• Gamma rays (from cobalt-60)
• approved used by U.S. FDA to process
chickens and red meat
• was used for food processing plants to
kill pathogens (such as Salmonella and
Campylobacter bacteria) in chickens
• the chickens are labeled “irradiated”
and marked with the green international
symbol for irradiation
ULTRASONIC WAVES
• are frequently used means of cleaning delicate
equipment in hospitals, medical clinics and dental
clinics
• Ultrasonic cleaners
• consists of tank filled with liquid solvent (usually
water) ; the short sound waves are then passed
through the liquid
• Sound waves – it mechanically dislodge organic
debris on instruments and glassware
FILTRATION
• Filters of various pore sizes
• are used to filter or separate cells, larger viruses, bacteria,
and certain other microorganisms from the liquids or gases
in which they are suspended
• Micropore filters – filters with tiny pore sizes that are
used in laboratories to filter bacteria and viruses out of
liquids
• High-efficiency particulate air (HEPA) filters
• a filter that is used to protect workers from contamination
• are located in operating rooms and patient rooms to filter
the air that enters or exits the room
GASEOUS ATMOSPHERE
• it is possible to inhibit the growth of
microorganisms by altering the atmosphere in
which they are located
• Gas gangrene – a deep wound infection that
causes rapid destruction of tissues
• caused by various anaerobes in the genus
Clostridium
USING CHEMICAL AGENTS TO INHIBIT
MICROBIAL GROWTH
• Disinfectants
• Antiseptics
DISINFECTANTS
• Chemical Disinfection
• refers to the use of chemical agents to
inhibit the growth of pathogens, either
temporarily or permanently
THESE FACTORS INCLUDE THE FOLLOWING:
• Prior cleaning of the object or surface to be disinfected
• The organic load that is present
• The bioburden
• The concentration of the disinfectant
• The contact time
• The physical nature of the object being disinfected
• Temperature and pH
CHARACTERISTICS OF AN IDEAL ANTIMICROBIAL
AGENT
• it should have a wide or broad antimicrobial spectrum
• it should be fast acting
• it should not be affected by the presence of organic matter (e.g. feces, blood, vomitus and pus)
• it must be nontoxic to human tissues and noncorrosive and nondestructive to materials on which it is used
• it should have residual antimicrobial film on the treated surface
• it must be soluble in water and easy to apply
• it should be inexpensive and easy to prepare, with simple, specific directions
• it must be stable both as a concentrate and as a working dilution, so that it can be shipped and stored for
reasonable periods
• it should be odorless
HOW DO DISINFECTANTS KILL MICROORGANISMS?
• Surface-active soaps & detergents, alcohols, phenolic compounds
• target and destroy cell membranes
• Halogens, hydrogen peroxide, salts of heavy metals, formaldehyde and
ethylene oxide
• destroy enzymes and structural proteins
PHENOL COEFFICIENT TEST
• To perform this test, a series of dilutions of phenol and the experimental disinfectant are
inoculated with the test bacteria, Salmonella typhi and S. aureus, at 370C
• the highest dilutions (lowest concentrations) that kill the bacteria after 10 minutes are
used to calculate phenol coefficient
ANTISEPTICS
• antimicrobial chemical agents that can safely be applied to skin
• reduces the number of organisms on a surface; it does not penetrate pores and hair
follicles to microorganism residing there
