Transcript Microbial Growth and Nutrition

Microbial Growth and
Nutrition
Nestor T. Hilvano, M.D., M.P.H.
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
Temperature Requirements
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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
• 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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2.
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
3. Differential – differentiates between organisms;
blood agar, levine EMB agar (lac+ =purple; lac=pink)
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
cont. variety of culture media
• On 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)
• 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
• Phases of microbial growth curve:
1. Lag phase – cells adjusting to environment, synthesize enzymes
2. Log phase- rapid growth & reproduction, increases logarithmically
3. Stationary phase- nutrients are depleted and waste accumulates,
rate of reproduction decreases; number of dying cells = number of
cells being produced
4. 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
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
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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.