1. Microbial
Growth

2. Optimum Temperature Requirements
Most human pathogens are ???

3. Physical Requirements
˚C- most bacteria destroyed by enzyme denaturation.
50-65 ˚C slow growth
15-50 ˚C Danger of food spoilage
5 -15 ˚C survival, and slow growth
0-5 ˚C slow or no growth
<0 ˚C no growth, but may survive.

4. Physical Requirements
<6.0 acidophiles
6 - 8 neutrophiles
>8.0 basophiles
Most pathogens are neutrophiles.

5. Physical Requirements
Osmotic Pressure
Isotonic % NaCl mOs
Hypotonic- low salt or pure water
Hypertonic - high salt - halophiles

6. Nutritional Classification
Photoautotroph – Energy = sunlight, Carbon source = CO2 Photosynthesis
Photoheterotroph - Energy source = sunlight, Carbon source = organic compounds.
Chemoautotrophs - Energy source = Oxidation of inorganic compounds Nitrates, Iron, Sulfur. Carbon source = CO2
Chemoheterotroph Both Energy and Carbon sources are organic compounds.

7. Chemical Requirements

8. Aerotolerance
Obligate Aerobe – Uses oxygen as final Hydrogen acceptor. Has Superoxide dismutase and catalase. Pseudomonas aeruginosa
Facultative Anaerobe – Can use O2, but other hydrogen acceptors i.e., nitrates. They are also capable of fermentation. Efficiency decreases without oxygen. Has Superoxide dismutase and catalase. E. coli, Staphylococcus sp.
Obligate Anaerobe – unable to use oxygen for energy. Most are harmed by it. No enzymes to detoxify oxygen forms. Clostridium sp.

9. Chemical Requirements
Anaerobe jar
Chemicals make CO2 and H2
H2 reacts with O2 + paladium --> H2O
O2 consumed.
Typical anaerobes:
Clostridium, Bacteroides

10. Aerotolerance continued
Aerotolerant Anaerobe – Cannot use oxygen i.e., Fermentative, but tolerate oxygen – possess Superoxide dismutase. Lactic acid producers.
Microaerophiles- Require oxygen but too much causes the production of toxic oxygen compounds. No enzymes. Lower levels of oxygen required for grow.
Capnophile – Some aerobic organisms require lower levels of oxygen and higher levels of CO2.
Neisseria meningiditis or gonorrhea

11. Special Techniques Anaerobic growth Extra CO2 -capnophilic -Neisseria
Selective media- selects for specific group.
Differential media-allows you to tell one group from another.

12. Toxic Forms of Oxygen Normal Atmospheric Molecular Oxygen O2
Most stable form of oxygen, but quite reactive.
Singlet oxygen – O single oxygen atom, higher energy state, much more reactive than O2. Found in phagocytes
Superoxide Free Radical O2- Produced during aerobic respiration must be detoxified by superoxide dismutase.
Peroxide anion O22- very reactive and toxic. Removed by catalase.

13. Chemical Requirements
Carbon -
Photosynthesis - Basis for all organic compounds
Nitrogen
From nitrates, ammoinum salts, amino acids, and nitrogen fixation
Needed for protein and nucleic acid synthesis
Phosphorous
From phosphate salts.
For DNA, RNA, ATP, etc
Sulfur
From sulfate ions, hydrogen sulfide and sulfur containing amino acids
For protein synthesis
Trace Elements
Mg, Fe, Mn, Zn, Cu etc for metabolism.

14. Culture Medium Medium Sterile - no living organisms present.
Simple - defined
Inoculum- microbes that start the culture
Culture = medium + microbes
Agar – polysaccharide derived from seaweed

15. Culture Medium Medium Agar - polysaccharide derived from seaweed
Chemically defined
Complex - serum, beef or yeast extract, peptones etc
Fastidious organisms have many nutritional requirements. Organic growth factors

16. Non-Selective Media Nutrient Agar Blood Agar LB agar (no antibiotics)
Tryptic Soy Agar –TSA in petri dishes
Nutrient Broth
Sugar tubes
TSA Agar (tube)
TSA broth
Brain Heart Infusion

17. Selective Media E. coli on EMB Mannitol Salt Agar Phenylethyl Alcohol
Inhibitor - Methylene blue
Indicator- Eosin
Gives a dark purple colony with green metallic sheen
Mannitol Salt Agar
Phenylethyl Alcohol
MacConkey Agar

18. Enrichment Media
Media to increase the numbers of desired species, but doesn’t purify them.
Used in soil and fecal samples.
Salmonella- Selenite broth.
Bacto-Dubos - M. tuberculosis
Grow overnight and then plate on elective medium.
Increases chances of isolation when numbers are very low or contaminants very high.

19. Obtaining Pure Cultures
Streak plate.
Each new direction diltues organisms until you get individual ones.
CFU
Serial dilution.
Dilute in broth until you get 1 organism. 1:10 --> 1:100 --> 1:000

20. Obtaining Pure Cultures

21. Preserving Cultures Large stock tubes - 4 to 6 months @ 4˚C
Freeze in medium with glycerol -20˚C- 1 year
Store -70˚C - 5 to 10 years
Store liquid nitrogen - indefinitely.
Lyophilization- freeze drying - indefinitely.

22. Frozen Culture Trick
Can’t thaw and freeze very many times- kills organisms.
Aseptically add mls of warm medium to top of the frozen vial.
Swish with pipette tip and remove liquid.
This thaws only a small top portion keeping the remainder frozen.
Can do this 10 times per vial.

23. Bacterial Growth Binary Fission one cell makes two
Cell first grows in size.
Replicates its DNA (genome)
Then divides.
Time it takes between divisions is the Generation Time.

24. Dynamics of Bacterial Growth
Generation Times GT.
E. coli - 20 minutes
Bacillus subtilis hours
Borrelia burgdorferi hours
Mycobacterium tuberculosis- 1 day
Treponema pallidum - 35 hours ( in rabbits)
Mycobacterium leprae - > 30 days

25. Phases of Growth Lag - no growth.
Exponential Growth- rapid growth- GT.
Stationary Phase- equal numbers dying and dividing.
Logarithmic Death - rapid death due to :
Lack of nutrients
Hostile media conditions (acid, toxic by-products)

26. Methods to Measure Growth
Direct
Plate Counts
Direct Counting
Indirect (estimations)
Turbidity
Metabolic activity
Dry Weight

27. Plate Counts
Perform serial dilutions
Plate out samples from dilutions

28. Plate Counts Can do spread plates for aerobes.
Can do pour plates for facultative anaerobes or microaerophiles.

29. Direct Counts Petroff-Hausser chamber Dilute sample.
Add volume of diluted medium to chamber.
Count bacteria
Calculate number in original sample.

30. Indirect Methods Metabolic Activity Dry Weight By product - CO2
More product / min = higher rate =
more bacteria
Dry Weight
Collect by centrifugation
Decant liquid
Dry
Weigh

31. Turbidity Use spectrophotometer.
Measure transmission in sterile medium.
Measure transmission in culture.