Module 3: Counting bacteria and microbe growth

CFU

colony forming unit

-term used when describing bacterial counts

ex: 1mL of sample caused 20 colonies to grow; 20 CFU/mL

Pour Plate Technique

1. Sample is added to empty petri dish

2. Sterile media/agar is added and mixed

3. Plate solidifies and is incubated

4. Colonies grow on and inside the agar

Spread plate technique

1. Sample is added to surface of agar plate

2. Sample is then spread over the surface of the agar using a sterile glass spreader

3. Plate is incubated

4. Colonies only grow on the surface

dilution

= volume of solution / total final volume

ex: 1mL of bacteria and 9mL of water = 1/10 dilution

serial dilutions

solutions being diluted multiple times to rapidly decrease concentration

standard plate count

-sample is serially diluted first and then plated

-30 to 300 colonies is the best range

standard plate count math

CFU/mL of original sample = colonies X reciprocal of dilution

ex: if 32 colonies are on plate 1/10,000 dilution, then the count is 32 X 10,000 which equals 32,000 CFU or 32 × 10 to the 4

lag phase

step in growth curve where bacteria are getting used to their environment

exponential phase

bacteria are growing quickly

-can determine generation time in this phase ( time it takes for bacteria to double in size)

stationary phase

broth is running low on nutrients and growth slows down

-rate of death= rate of division

death phase

no nutrients; bacteria begin to die

turbidity

is measured using spectrophotometry

-a wavelength of light is passed through the sample

-high turbidity= less light passes through

-high turbidity= higher optical density

spectrophotometer

set to 550-600 nm for bacteria

-bacterial sample is placed into small rectangular cuvette that is then place in the device

psychrophiles

organisms that really like the cold

-optimum temperature is 0-5 C

psychotroph

optimum temperature is 20 C

• like the cold but not too cold

mesophiles

optimum is 37 C

thermophiles

optimum is 70 C

extreme thermophiles

optimum temp is 120-125 C

thioglycolate tubes

used to measure oxygen growth requirements

-oxygen is present at the top, red part of the tube

-oxygen is absent at the bottom of the tube

strict aerobe

needs oxygen to survive

-found at the top of thioglycolate tubes

strict anaerobes

can’t survive in presence of oxygen

-found at bottom of thioglycolate tubes

facultative anaerobes

can live with or without oxygen, but prefers oxygen

-found throughout the thioglycolate tubes but is slightly more concentrated at the top

microaerophile

likes only some oxygen

-grows right under the top layer of the thioglycolate tubes

aerotolerant

grows the same with or without oxygen

-evenly distributed throughout the thioglycolate tubes

gas pack jar

airtight jar used to create an anaerobic environment

-used to grow petri dishes with microbes that are strict anaerobes

acidophiles

optimum pH below 5.5

neutrophiles

optimum pH near 7

alkalinophiles

optimum pH above 8

halotolerant

microbes that can grow in high salt environments

halophile

microbes that require high salt environments above 13% salt concentration

osmophile

can grow in high sugar environements