Download Final Microbial Physiology

INTRODUCTION TO FOOD SAFETY
AND QUALITY MANAGEMENT
CURRICULUM (M. Phil) Program at FCC
Dr. Quratulain Syed
Chief Scientific Officer
Food & Biotechnology Research Centre,
PCSIR, Labs, Complex ,Lahore
Course : Food Microbiology &
Toxicology
Credit Hours:
3(2-1)
Objectives:
• To understand different microbial threats
related to food safety and epidemiology of
different food borne illness.
• To understand the international microbial
limits for safe foods, toxicological aspect of
foods and their impact
• Food microbiology including microbial analysis,
growth, physiology and survival;
• Interpret the Codex & ICMSF microbiological
criteria regulations for foodstuff;
• Role and ability of antimicrobial agents and their role
in cleaning science;
• Comprehend results relating to the detection,
enumeration, identification and prediction of microorganisms; explain the etiology of common food
borne illnesses/diseases;
• Role of microbes in disease, food spoilage, food
production,
• Food preservation methods and biotechnology;
• Importance of water quality, water chlorination
(chemistry, methods, testing and interpretation of
results) in food production.
• Food toxicology: Overview - intrinsic and extraneous
toxins; Principles, types, branches; Toxicity: curve,
factors influencing potency, margin of safety, factors
influencing toxicity; Dose-response relationship,
manifestation of organ toxicity.
• Measurement of toxicants and toxicity;
Toxicokinetics: carcinogenesis, mutagenesis,
teratogensis;
Practicals
• Safety in microbiological laboratory.
• Basic functions and handling of laboratory
equipments. Use of microscope.
• Sterilization and disinfection of glassware.
• Preparation of culture media. Staining of
microorganisms and their structures.
• Bacterial cultivation, growth measurement.
Characteristics of bacterial colonies. Bacterial
and fungal morphology. Micrometry.
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Microbial physiology
6
The Domain of Life
Comparative ribosomal RNA sequencing has defined
the three domains of life:
• Bacteria
•Archaea
•Eukarya
Prokaryotic Diversity
• Several lineages are present in the domains
Bacteria and Archaea
• Enormous diversity of cell morphologies
and physiologies
Cell Structure
•
Two structural types of cells are recognized:
the prokaryote and the eukaryote.
•
Prokaryotic cells have a simpler internal
structure
than
eukaryotic
cells,
membrane-enclosed organelles.
lacking
Bacterial Morphology
Some typical bacterial morphologies include
• Coccus,
• Rod / Bacilli
• Coccobacilli
• Spirillum,
• Spirochete,
• Appendaged,
• Filamentous.
Cytoplasmic Membrane
The cytoplasmic membrane
• Highly selective permeability barrier
• lipids and proteins
• Forms a bilayer with hydrophilic exteriors and a
hydrophobic interior.
Cell Wall
• Gram-negative Bacteria have only a few layers of
peptidoglycan , but gram-positive Bacteria have
several layers.
• In addition to peptidoglycan, gram-negative
Bacteria contain an outer membrane consisting of
lipo-polysaccharide (LPS), protein, and lipo-protein.
Cell wall
• Surrounds the cyptoplasmic membrane
• Directly reflects adaptive strategies involved with
-Uptake of nutrients
- Excretion of waste products
-Movements
-Protection
-Adhesion
• In some organisms >25 of the genome is devoted
to its synthesis, regulation and maintenance
Gram positive cell wall:
• Rigid structure
• Based on a crossed-linked polymer
-Peptidoglycan
• Also contains teichoic acids (2 types)
-Wall teichoic acids
 Polymer consisting of ribitol and phosphate
 Confer antigenic specificity for the bacteria
Gram negative membrane:
• Consist of outer and inner (cytoplasmic) membrane
separated by the periplasm
• Outer membrane
- Flexible outer phospholipid bilayer with an inner
peptidoglycan layer
 Strong negative charge of phospholipid bilayer helps evade
phagocytosis
 Also protects against some antibiotics
- Outer membrane also contain hydrophobic
lipopolysaccharides
and lipoproteins
• Periplasm:
- Solution between the inner and ouetr membrane
- Contains specific periplasmic proteins
 Usually involved in hydrolysis and transport of materials
• Cytoplasmic (inner) membrane:
- Feature of both Gram-positive and Gram-negative
cells
- Phospholipid bilayer
- Allow the pasage of membrane components through
- Has peripheral or integral proteins associated with it
Chemically, bacteria consist of:
• Water (75-85%) –
bound water and
free water
• Dry matter (1525%) – organic
part and mineral
substances
(inorganic part)
Dry matter
• Organic part
• proteins – 50-80%
• nucleic acid – 10-30%
• carbohydrates – 12-18%
• polysaccharides – 3-5%
• lipids – 5-10%.
• Inorganic part
• nitrogen (N), carbon (C),
oxygen (O), hydrogen (H),
phosphorus (P), sulfur (S),
sodium (Na), magnesium
(Mg), potassium (K),
calcium (Ca), iron (Fe)
and other
Growth of Microbes
• Increase in number of
cells, not cell size
• One cell becomes
colony of millions of
cells
Growth of Microbes
• Control of growth is important for
– Infection control
– Growth of industrial products and biotech
organisms
Cell Growth
• Microbial growth involves an increase in the
number of cells. Growth of most microorganisms
occurs by the process of binary fission.
•
Alternative means
Budding
Conidiospores (filamentous bacteria)
Fragmentation
Generation Time
• Time required for cell to divide/for
population to double
• Average for bacteria is 1-3 hours
• E. coli generation time = 20 min
– 20 generations (7 hours), 1 cell becomes 1 million cells!
Fig. 7.14a
Growth and survival
38
Standard Growth Curve
Phases of Growth
• Lag phase: adaptation to the environment making new
enzymes in response to new medium
• Exponential logarithmic growth: Desired for production
of products . Most sensitive to drugs and radiation during
this period
• Stationary phase: nutrition exhausted, toxin increased
– death rate = division rate
• Decline: cell die (steady biomass) or lysis (decrease
biomass) death exceeds division
• Dormant as spore, non-viable state
Measuring Growth
• Direct methods – count individual cells
• Indirect Methods – measure effects of
bacterial growth
Measuring Microbial Growth
There are 4 surrogate ways:
• Cell counting (direct),
•
Colony formation (direct) ,
•
Biomass determination (indirect),
• Turbidity (indirect)
• Microbes are useful tools in research because of their rapid
life cycle, their simple growth requirements, and their
small size.
Growth measurement
•
Cell count: microscopic
observation; flow
cytometer (direct)
•
Colony formation:
Measure the living cell
(direct)
•
Biomass determination:
dry weight; essential cell
component (indirect)
•
Turbidity (indirect)
Counting the viable cells: Dilution
Agar : Solidifying medium
Indirect Counting
• Turbidity measurements are an indirect but very
rapid and useful method of measuring microbial
growth. However, to relate a direct cell count to a
turbidity value, a standard curve must first be
established.
Turbidity
Metabolic Activity
Dry Weight
Factors Regulating Growth
• Nutrients
• Environmental
conditions:
• Generation time
Nutrients (Chemical Requirements)
• Water
• Elements
– C (50% of cell’s dry weight)
– Trace elements
• Organic
– Source of energy (glucose)
– Vitamins (coenzymes)
– Some amino acids, purines and pyrimidines
Water
• Microbes require water to dissolve enzymes and
nutrients required in metabolism
• Water is important reactant in many metabolic reactions
• Most cells die in absence of water
– Some have cell walls that retain water
– Endospores and cysts cease most metabolic activity in a dry
environment for years
• Two physical effects of water
– Osmotic pressure
– Hydrostatic pressure
Osmotic Pressure
• Is the pressure exerted on a semi-permeable membrane
by a solution containing solutes that cannot freely cross
membrane; related to concentration of dissolved
molecules and ions in a solution
• Hypotonic solutions have lower solute concentrations;
cells placed in these solutions will swell and burst
Osmotic Pressure
• Hypertonic solutions have greater solute
concentrations; cells placed in these solutions will
undergo cremation (shriveling of cytoplasm)
– This effect helps preserve some foods
• Restricts organisms to certain environments
– Obligate halophiles – grow in up to 30% salt
– Facultative halophiles – can tolerate high salt
concentrations
Hydrostatic Pressure
• Water exerts pressure in proportion to its depth
– For every addition of depth, water pressure increases
1 atm
• Organisms that live under extreme pressure are
barophiles
– Their membranes and enzymes depend on this
pressure to maintain their three-dimensional,
functional shape
Environmental Effects on
Microbial Growth
Factors affecting Growth
• The orderly increase in the sum of all the components
of an organism
Affected by:
 Nutrients
 pH: neutrophils, acidophils, alkalophils
 Temperature: psychrophils; mesophils; themophils,
thermodurics
 Aeration
 Pressure
 Ionic strength and osmotic pressure: halophils, osmophils
1. Temperature
• Temperature is a major environmental factor
controlling microbial growth.
• The cardinal temperatures
- minimum, optimum, and maximum.
• Microorganisms can be grouped by the
temperature ranges they require.
2. Low or High pH
• The acidity or alkalinity of an environment
can greatly affect microbial growth.
• Organisms that grow best at low pH are
called acidophiles; those that grow best at
high pH are called alkaliphiles.
• Some organisms have evolved to grow best at low
or high pH, but most organisms grow best between
pH 6 and 8.
• The internal pH of a cell must stay relatively close
to neutral even though the external pH is highly
acidic or basic.
• Acid (below pH 4) good preservative for pickles,
cheese
pH
• Many bacteria and viruses
survive low pH of stomach to
infect intestines
• Helicobacter pylori lives in
stomach under mucus layer
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3. Air requirement:O2

Aerobe: A microorganism whose growth requires
the presence of air or free oxygen

Anaerobe: A microorganism that grows only or
best in the absence of free oxygen. Organisms
utilize bound oxygen

Microaerophile: A microorganism that grows best
in the presence of low concentrations of oxygen

Facultative anaerobe/aerobe: A microbe
that adjusts its metabolism to depending on
the oxygen concentration in which it is
growing. can live with or without oxygen.

Aerotolerant anaerobe: an organism that always
grows in an anaerobic mode -- can tolerate oxygen
and grow in its presence even though they cannot
use it.

Capneic microbe: An organism that requires 3 to
10% CO2 for growth
Growth pattern
1.
2.
3.
4.
5.
Obligate aerobe
Obligate anaerobe
Microaerophile
Aerotolerant anaerobe
Facultative anaerobe/aerobe
Two types of biofilms
when cells grow as biofilm
Environmental
Disease-associated
 Symbioses
 Termite, ruminant




digestion
Sewage treatment
bioreactors
Water pipes
Dental units
Contact lens cases
 Dental plaque
 Endocarditis
 Cystic Fibrosis
 Otitis media
 Urinary catheter
 Implants
• Special
techniques are
needed to grow
aerobic and
anaerobic
microorganisms.
• Several toxic forms of oxygen can be formed in
the cell as the result of respiration, but enzymes
are present that can neutralize most of them.
Hydrogen peroxide is one of those forms that
can be neutralized by catalase.
4. Salinity
• Some microorganisms (halophiles) have
evolved to grow best at reduced water
potential, and some (extreme halophiles)
even require high levels of salts for growth.
extreme halophiles can live in solutions of
25 % salt; seawater = 2% salt
Extremophiles
• Definition - Lover of extremities
• Temperature extremes
– Boiling or freezing, 1000C to -10C
– Chemical extremes
– Vinegar or ammonia (<5 pH or >9 pH)
– Highly saline, up to x10 sea water
• we sterilize & preserve foods today
Extreme Temperatures
• Thermophiles - High temperature
– Thermal vents and hot springs
– May go hand in hand with chemical extremes
• Psychrophiles - Low temperature
– Arctic and Antarctic
•
1/2 of earth’s surface is oceans between 1-40C
•
Deep sea –10C to 40C
•
Most rely on photosynthesis
Chemical Extremes
• Acidophiles - Acidic
– Again some thermal vents & hot springs
• Alkaliphiles - Alkaline
– Soda lakes in Africa and Western U.S.
• Halophiles - Highly saline
– Natural salt lakes and manmade pools
Extremozymes
• Enzyme from Extremophile
– Industry & Medicine
• What if you want an enzyme to work
– In a hot factory?
– Tank of cold solution?
– Acidic pond?
– Sewage (ammonia)?
– Highly saline solution?
– Pay a genetic engineer to design a “super” enzymes...
– Extremophiles have the enzymes that work in extreme
conditions
“Compatible Solute” Strategy
• Cells maintain low concentrations of salt in their
cytoplasm by balancing osmotic potential with organic,
compatible solutes.
• They do this by the synthesis or uptake of compatible
solutes- glycerol, sugars and their derivatives, amino acids
and their derivatives & quaternary amines such as glycine
betaine.
• Energetically synthesizing solutes is an expensive process.
– Autotrophs use between 30 to 90 molecules of ATP to
synthesize one molecule of compatible solute.
– Heterotrophs use between 23 to 79 ATP.
Osmoregulation
• Halophiles have adapted to life at high salinity
in many different ways.
– Structural modification of external cell walls- posses
negatively charged proteins on the outside which bind
to positively charged sodium ions in their external
environments & stabilizes the cell wall break down.
Halophiles
• The “salt-in” strategy uses less energy but requires
intracellular adaptations. Only a few prokaryotes
use it.
• All other halophiles use the “compatible solute”
strategy that is energy expensive but does not
require special adaptations.
Mean Generation Time
and Growth Rate
• The mean generation time (doubling time) is
the amount of time required for the
concentration of cells to double during the
log stage. It is expressed in units of minutes.
1
• Growth rate (min-1) =mean generation time
• Mean generation time can be determined
directly from a semilog plot of bacterial
concentration vs time after inoculation
Classification of bacteria based on
nutritional requirements
• Autotrophs are free-living, most of which can use
carbon dioxide as their carbon source. The energy can
be obtained from:
• sunlight – protoautotrophs (get energy from
photochemical reactions)
• inorganic
compounds,
by
oxidation
–
chemoautotrophs (get energy from chemical
reactions)
• Heterotrophs are generally parasitic bacteria,
requiring more complex organic compounds than
carbon dioxide, e.g. sugars, as their source of carbon
and energy.
Methods of laboratory diagnosis
1.
2.
3.
4.
5.
6.
Bacterioscopical (Microscopic examination)
Bacteriological (Culture method)
Detection sensitivity of bacteria to antibiotics
Serological
Biological
DNA-technology test (PCR)
In the clinical laboratory it is necessary:
• isolate bacteria in pure culture;
• obtain
sufficient
growth
of
bacteria
for
demonstration their properties such as study of
morphological, cultural, biochemical, antigenic and
pathogenic properties, bacteriophage and bacteriocin
susceptibility;
• determine a sensitivity
to antibiotics.
Methods of the cultivation
•
Streak culture (surface plating). The inoculum is spreaded
thinly over the plate of a culture media in series of parallel
lines in different segment of the plate. On inoculation well
separated colonies are obtained over the final series of
streaks.
Methods of the cultivation
• Lawn or carpet culture. Lawn cultures are prepared by
flooding the surface or plate with suspension of bacteria. It
provides uniform surface growth of bacteria. It is useful for
bacteriophage typing and antibiotic sensitivity test.
Methods of the cultivation
• Stroke culture. It is made in tubes containing agar
slopes. It is used for providing a pure growth of
bacterium (for slide agglutination).
Methods of the cultivation
• Stab culture. It is prepared by puncturing with
charged long straight wire (loop). Stab culture is
employed mainly for cultivation of anaerobes.
Pure plate culture
•
Methods
of
the
cultivation
Liquid culture in a tube, bottle or flask may be
inoculated by touching with a charged loop
•
Identification
of
bacteria
Microscopic examination: It helps to detect a shape, a size
and an arrangement of microorganisms
• Staining reaction: On gram staining we can have two groups
of microorganisms: Gram positive and Gram negative.
E. coli, Gram negative (A), Staphylococcus epidermidis,
Gram positive (B) and Bacillus cereus, Gram positive
•
Identification
of
bacteria
Motility: Some bacteria can move (Salmonella, E. coli,
Proteus, Pseudomonas, Vibrions, Clostridia). Dark
ground microscopy and Phase contrast microscopy,
special culture media use for studying motility of
bacteria
Special stain for flagella
Identification of bacteria
• Culture character: Growth requirement, colonial characteristics
in culture
Colony morphology descriptions
Colony morphology
•
Identification of bacteria
Metabolism: Capacity to form pigment and power of haemolysis
is help for classification of bacteria
Staphylococcus
aureus
Micrococcus
roseus
Studying of
haemolysis
Colonies and pigments of bacteria
•
Identification
of
bacteria
Biochemical reactions: The more important
widely used tests are as under:
• a) Sugar fermentation
and
•
Identification
of
bacteria
b) Indole production
• c) Hydrogen sulfide production
Identification of bacteria
• d) Other tests: Citrate utilization; Nitrate reduction; Methyl
red test; Urease test; Catalase test; Oxidase reactions.
Positive Catalase Test
on Staphylococcus aureus
Negative Catalase Test
on Streptococcus lactis
API-20 "Bio Merieux" (France) strip test
• Twenty tests are performed on this strip by a simple
procedure, saving time and money.
Escherichia
coli
Enterobacter
agglomerans
Edwardsiella
hoshinae
Identification of bacteria
• Antigenic analysis: by using specific sera we can
identify microorganism by agglutination reaction
(Serologic Typing of Shigella).
The clumping of the bacteria
is seen in this circle
No clumping of the bacteria
is seen in this circle
•
Identification
of
bacteria
Pathogenicity: For pathogenicity test commonly
used
laboratory animal models are guinea pig, rabbit, rat and
mouse.
• Resistance to antibiotics and other agents
• Metabolism is the process
of building up chemical
compounds in the cell and
their breaking down during
activity to receive the
required energy and the
building elements.
• Metabolism comprises of
anabolism
(assimilation)
and
catabolism
(dissimilation)
•
•
•
•
Classification of Media
A. On the basic of consistency:
Solid media
Liquid media
Semisolid media
Classification of Media
• Nutrient media can be subdivided:
• 1. Simple media - meat-peptone broth (MPB),
meat-peptone agar (MPA)
• 2. Synthetic media
• 3. Complex media
• 4. Special media: a) Enriched media; b)
Enrichment media; c) Selective media; d)
Indicator and differential media; e) Sugar media;
f) Transport media.
• 5. Aerobic and anaerobic media according to type of respiration bacteria
subdivided into 4 groups:
• Obligate aerobes (Brucella)
• Microaerophils (H.pylori)
• Obligate Anaerobes (C.tetani)
• Facultative Anaerobes (E.coli)
Sterilization
TREATMENT
Incineration
TEMPERATURE
>500 C
EFFECTIVENESS
Boiling
100 C
Thirty minutes of boiling kills vegetative
forms of bacteria but may not kill bacterial
endospores. There are also toxins that are not
inactivated at 100C.
Intermittent
boiling
100 C
Three 30-minute intervals of boiling,
followed by periods of cooling kills bacterial
endospores.
Vaporizes organic material on nonflammable
surfaces but may destroy many substances in
the process.
Autoclave
121 C for 15 Kills all forms of life including bacterial
(steam under minutes at 15 endospores. The substance being sterilized
pressure)
p.s.i.
must be maintained at the effective
temperature for the entire time.
Sterilization
Dry heat
(hot air
oven)
160 C for 2
hours
Used for materials that must remain dry.
Good for glassware, metal, but not most
plastic or rubber items.
Dry heat
(hot air
oven)
170 C for 1
hour
Same as above. Note that increasing the
temperature by 10 C shortens the sterilizing
time by 50 %.
Pasteurizatio
n (batch
method)
63-66 C for
30 minutes
Kills most vegetative bacterial cells,
including pathogens such as streptococci,
staphylococci
and
Mycobacterium
tuberculosis.
Pasteurizatio
n (flash
method)
72 C for 15
seconds
Effect on bacterial cells is similar to batch
method. For milk, this method has fewer
undesirable effects on quality or taste.
AUTOCLAVES (1) AND HOT AIR OVEN (2)
1
2