Transcript 07 Microbial Growth and Nutrition

Microbial Growth and
Nutrition
Nestor T. Hilvano, M.D., M.P.H.
Images Copyright by Bauman, Robert. 2009. Microbiology, With
Diseases by Taxonomy, 3rd edition, Pearson Benjamin Cummings
Learning Objectives
You should be able to:
1. Compare the four basic categories of organisms based on
their carbon and energy sources.
2. Distinguish among anaerobes, aerobes, aerotolerant
anaerobes, facultative anaerobes, and microaerophiles.
3. Explain how extremes of temperature, pH, and osmotic
pressure limit microbial growth.
4. Describe methods for collecting clinical specimens.
5. Describe the two most common methods by which
microorganisms can be isolated for culture.
6. Describe six types of general culture media available for
bacterial culture.
7. Explain what is meant by the generation time of bacteria.
8. Draw, label, and describe a bacterial growth curve.
9. Contrast viable plate count and turbidity methods of measuring
bacterial growth.
Microbial Growth Requirements
• Chemical
1. Nutrients – C,O, N, H; source of carbon,
energy, and electrons or hydrogen atoms
2. Trace elements and organic materials,
growth factors
• Physical – temperature, pH, osmolarity
and pressure
Carbon and Energy Sources
• Photoautotrophs – use
CO2 & energy from sunlight
• Chemoautotrophs – use
CO2 & energy from organic
cpds.
• Photoheterotrophs – use
organic cpds. & energy from
sunlight
• Chemoheterotrophs – use
carbon and energy from same
source (sugars, lipids,
proteins, etc.)
Oxygen Requirements
• Aerobes (obligate aerobes) –
need O2.
• Anaerobes – w/o O2
1. obligate anaerobes –
can’t tolerate O2
2. Facultative anaerobes –
live with or without O2, can
ferment; ex. E. coli
3. Aerotolerant anaerobes –
does not use O2, O2 does not
harm them; ex. lactobacilli
• Microaerophiles – require low
level of O2; ex. Helecobacter pylori
Other Chemical Requirements
• Nitrogen - Growth limiting nutrient, from
organic (proteins, amino acids, DNA) and
inorganic (metabolic waste); some
bacteria can reduce nitrogen gas into
ammonia (nitrogen fixation)
• Trace elements – selenium, zinc, copper,
magnesium, iron, manganese, phosphorus
• Growth factors - vitamins, cofactors,
coenzymes, essential amino acids;
required by fastidious organisms
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Temperature Requirements
Optimum temperature
Psychrophiles (-8˚C- 18˚C),
Psychrotrophs (0˚C – 32˚C; food spoilers)
Mesophiles (20-40˚C; human pathogens)
Thermophiles (>45˚C; hot springs & compost piles)
Hyperthermophiles (>80˚C; hot vents)
pH, Atmospheric and Osmotic Pressure
• pH
- neutrophiles (optimum pH 6.5 – 7.5; human
pathogens)
- acidophiles (pH <4)
- alkalinophiles (pH >8)
• Atmospheric pressure – 760 mmHg
• Osmotic pressure (280-300 mOsm)
- halophiles (salt loving); obligate halophiles (need
high salt); facultative halophiles (don’t require but
can tolerate salty conditions)
Culturing Microorganisms
• Clinical specimen –
feces, saliva, CSF, urine,
blood, skin, mucous
membrane, discharge or
diseases tissue.
• Culture – microbes that
grow from an inoculum
• Obtaining pure culture
(progenitor, CFU)
- aseptic technique
Pure Culture: Streak Plate Method
• Spread an inoculum (0.1 ml. on top) using sterile
inoculating loop across the surface of solid
medium in petri dish; incubate
Pure Culture: Pour Plate Method
• CFUs separated from each other using a
serial dilutions. Final dilutions are mixed w/
warm agar in petri dish
Culture Media
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1.
2.
3.
Nutrient broth, agar = complex polysaccharide
6 variety of culture media:
Defined – exact composition is known
Complex – contain variety of growth factors and can
support a wider variety of microbes; TSA, nutrient broth
Differential – differentiates between organisms; blood
agar, levine EMB agar (lac+ =purple; lac- =pink)
BLOOD AGAR (BAP)
Strep. Pyogenes – beta
hemolysis (clear yellowish
zone, complete)
Strep. Pneumoniae – alpha
hemolysis (greenish halo,
partial)
Enterococcus – gamma
hemolysis (no red cells
hemolyzed, no change
around medium)
E. Coli fermented sugar, produced
acid (turn red phenol to yellow) and
produced gas (bubble)
cont. variety of culture media
4. Selective – contain substance that either favor or
inhibit microbial growth; sabouraud dextrose agar
(slightly low pH is selective for fungi); Mc Conkey
agar selective for gm. - and lactose fermenting
Nutrient agar
Sabouraud dextrose agar
McConkey agar = selective for gm. –
E. coli and differentiates lactose
Fermenting E. coli (red to pink colonies)
cont. variety of culture media
5. Reducing
(anaerobic) –
conducive to
anaerobes, contain
sodium thioglycollate,
that combine with
free O2 and remove
it.
6. Transport – to carry
clinical specimens
a) gas-pak anaerobic system
b) anaerobic glove box
Microbial Growth
• Logarithmic (exponential) growth – binary fission,
greater yields than simple addition
• Generation time – time required for a pop. of
cells to double in number
arithmetic graph
Semilog graph
Phases of Microbial Growth Curve
• Lag phase – cells adjusting to
environment, synthesize enzymes
• Log phase- rapid growth &
reproduction, increases logarithmically
• Stationary phase- nutrients are
depleted and waste accumulates, rate of
reproduction decreases; number of dying
cells = number of cells being produced
• Death phase- cells die at faster rate
than number of new cells produced
•
Measuring Microbial Growth
Direct Methods
a. viable plate counts
- make serial dilutions; count #
colonies on spread or pour plate
from each dilution
b. membrane filtration
- large sample poured thru filter
to trap cells, transferred to solid
media and colonies counted
c. microscopic count
- sample placed on cell counter
and viewed thru microscope
d. electronic counter- device that
counts cells as they interrupt an
electrical current
e. most probable number (MPN) –
statistical estimate; the more
bacteria in a sample, the more
dilutions are required to reduce
their # to zero
Viable plate count
Measuring Microbial Growth
• Indirect method
a) turbidity - use of
spectrophotometer; a
more turbid broth
culture will have a
greater population
b) metabolic activity measure nutrients,
wastes
c) dry weight –
oragnisms are filtered
from culture medium,
dried, and weighed
Homework
1.
2.
3.
4.
5.
6.
7.
Define terms: autotrophs, heterotrophs, aerobes,
anaerobes, facultative anaerobes, thermophiles,
psychrotrophs, halophiles, inoculum, culture,
generation time, and logarithmic curve.
Describe blood agar as differential culture for gamma,
beta and alpha hemolysis of organism.
What culture medium is selective for gram – and
lactose fermenting E. coli?
Describe measuring bacterial growth by viable plate
count and turbidity methods.
Discuss at least 3 physical and chemical requirements
for microbial growth in the laboratory.
Discuss the phases of microbial growth curve.
List clinical specimens used in doing microbial culture.