Micro Lab 6 - Microbial Growth Requirements (last for exam 1)

Ocean Floor Prokaryotes

very low temperatures (about 4°C)

Geothermal vents Prokaryotes

very high temperatures up to 400°C) (very high atmospheric pressure- up to 1000x sea level.

Salt lakes Prokaryotes

up to 13% salt

Geysers

about 95°C

Psychrophiles

0.5- 20 °C

-very cold

-LOW fluidity, proteins rigid, slow diffusion

-unsaturated

-HIGH hydrophobicity

-HIGH flexabiliity

Mesophiles

20-45 °C

-moderate temp

Thermophiles

50-80 °C

-high temp

-membrane too fluid and therefore impairing metabolism; proteins + nucleic acids denature

Hyperthermophiles

80-110°C

-evolbes/adapts

-very high temp

-saturated

-HIGH bonds and prevents denaturation

Halotolerant

negative numbers - 10% NACI

-doesnt require salt for growth, but can grow in high salts

Nonhalophiles

less than 1% NACI

-grows best in low salt concentrations

Halophile

0-12% NACI

-thrives in high salt concentrations

Slight Halophiles

1.7-4.8% NACI

Moderate Halophiles

4.7-20% NACI

Extreme halophile

20-30% NACI

-very high salt concentrations needed to grow

Radiation

Some microbes are capable of withstanding radiation through the formation of endospores. These organisms often can live for longer periods of UV exposure than non-spore forming organisms.

Anaerobic organisms

must be handled in a way so as not to expose them to oxygen from the time a culture is taken from a patient until they are grown in the lab.

Special anaerobic sample devices

Chemical packet = when opened, consumes the oxygen to near zero level and they often also release CO2 which stimulates the growth of some anaerobes.

Oxygen indicator = turns color when oxygen is present. Many obligate anaerobes will grow on the same media as aerobic bacteria.

Tryptic soy agar for both the aerobic and anaerobic incubations

Bacteria being used

Serratia marcescens

Staphylococcus epidermidis

Escherichia coli

Bacillus subtilis

Activity 1: Determining the Optimal Temperature for Growth of Bacteria

Inoculate 5 broths with assigned microorganism.

Incubate one each at 5°C, 25°C, 37°C, 45°C, and 60°C and leave one uninoculated

turbidity

can be described as: none, slight, medium, or heavy.

Activity 2: Determining the Optimal Osmotic Pressure for Growth of Bacteria

Inoculate each salt broth with assigned microorganism.

Incubate at 37°C for 24-48h

Activity 3: UV radiation

Swab 4 plates with assigned organism. Swab the plate for confluent growth.

One plate will act as your control, do not expose to UV.

For the other 3 plates, expose them to UV for the following times -1m, 2.5m, or 5m.

-During exposure the lids should be off, UV light is impeded by the plastic covers.

-It is easiest to put all 3 plates under the UV lamp and start your 5 min timer. At 1 min remove the first plate, at 2.5 min remove the second, and remove the third when the timer goes off.

After exposure, cover the plates, and incubate at 37°C for 24-48h

Growth can be

· None

· Slight - few colonies

· Moderate - confluents but some areas with no growth

· Heavy - confluent

Activity 4: Growing microbes in anaerobic bags.

Pseudomonas aeruginosa

Escherichia coli

Enterococcus faecalis

Clostridium sporogenes

2 TSA plates per group of 4 students

Each student works with one species from the list above.

Students working in groups of 4 will streak their organism in 1 quadrant of the agar plate.

One plate will be incubated aerobically at 37ºC in a canister and 1 plate will be incubated at 37C in an anaerobic bag until next lab.

Activity 5: Growing microbes in anaerobic media

Each student will need one tube of thioglycolate media.

Inoculate the thioglycolate media with the bacteria chosen in Activity 4.

Incubate at 37°C for 24-48h