Chapter 6- Microbial Growth

Question 1

purple/green sulfur bacteria

Answer 1

photoautotrophs that are anoxygenic that use light as a primary source of energy, but then use sulfur to convert CO2 to sugar

Question 2

purple/green nonsulfur bacteria

Answer 2

Use light as primary source of energy, but use organic compounds to reduce CO2.

Question 3

What distinguishes purple/green sulfur bacteria from each other?

Answer 3

green- chlorophyll located in vesicles (chlorosomes) underlying and attached to plasma membrane

purple- located in invaginations (chromatophores).

location of stored sulfur

16S rRNA sequence

Question 4

Harmful Oxygen species

Answer 4

Singlet- O2- excited form of O2 that has an e- in a higher orbital due to energy absorption

superoxide free radicals- O2^1- : formed from respiration; can be neutralized to oxygen and hydrogen peroxide by superoxide dismutase in organisms that can at least tolerate oxygenated conditions

peroxide anion- O2^2-: comes from hydrogen peroxide. catalase can convert hydrogen peroxide into water and oxygen, while peroxidase can convert it to water. produced from respiration

hydroxyl radical- OH*: formed by ionizing radiation and respiration in traces; THE MOST REACTIVE

Question 5

Which organisms have catalase/ superoxide dismutase

Answer 5

Obligate aerobe - both
Facultative anaerobe - both
Obligate anaerobe - neither
Aerotolerant anaerobe - only SOD
Microaerophiles - SOD+ catalase +/- (small amounts

Question 6

Biofilm characteristics

Answer 6

3-D structure not well appreciated until the development of confocal microscopy

usually attached to a surface, may facilitate conjugation, works cooperatively, sheltered from desiccation, antibiotics and the immune system, 10-1000x’s resistant to antimicrobials.

Question 7

Quorum sensing

Answer 7

the ability of bacteria to coordinate gene expression with other bacteria using signaling molecules; usually respond to local population density. coordinates genes for surfactants, individual/group growth, and biofilm dispersion.

Question 8

How can you prevent biofilm formation?

Answer 8

Block chemical signals, incoporate antimicrobials into surfaces, use lactoferrin to stimulate surface motility and prevent formation.

Question 9

Soft agar and solid agar

Answer 9

Soft agar: less than a 1% agar solution, used to test for motility

solid agar: 1.5-1.8%, regular culture

Question 10

Ways to remove O2 from media prior to inoculation?

Answer 10

heating- liquid media can be heated to drive-off dissolved O2 then you can seal it.

reducing media- using a chemical like thioglycolate reduces O2 to water.

incubating a plate in an oxygen free environment (anaerobic jar with bicarbonate, borohydride and Pd catalyst pellets) *this is useful if brief exposure to oxygen isn’t fatal.

incorporating oxyrase into media (useful if brief O2 exposure isn’t lethal).

Question 11

Capnophiles

Answer 11

Grow better at high CO2 concentrations (because of the similarity to the intestines, respiratory tract, and other tissues)

Question 12

Dextrose agar

Answer 12

selective media that encourages growth of fungi by acidic pH of 5.6 and the presence of chloramphenicol, which is an antibiotic that targets prokaryotes.

Question 13

Bismuth agar

Answer 13

selective/differential medium that differentiates Salmonella species, usually S. typhi from feces. Bismuth inhibits gram + bacteria and most gram - bacteria except Salmonella.

*broth also contains ferrous sulfate because S.typhi is the only Salmonella species that reduces it to hydrogen sulfide. *

Question 14

Blood agar

Answer 14

distinguishes bacteria that lyse RBCs (ex. S. pyogenes)

Question 15

mannitol salt agar

Answer 15

used to select for/distinguish S.aureus

has high salt 7.5% NaCl to discourage growth of other organisms, and has a pH indicator that changes color when mannitol is fermented.

Question 16

MacConkey Agar

Answer 16

distinguishes and selects for Gram - cells that ferment lactose.

Bile salts and CV discourage growth of Gram +, lactose + pH indicator are for lactose fementers that will produce pink or red colonies.

Question 17

Enrichment culture

Answer 17

Used to favor the growth of an organism, designed to increase very small levels to detectable levels (usually done in liquid and for fecal/soil samples). Example: boiling a culture to get Clostridium endospores, or isolating for a bacterium that can metabolize phenol.

Question 18

preserving bacterial cultures

Answer 18

days-weeks: refrigeration

years:
- deep freezing -80 C or - lyophilization (-54 to -72 C ) then sublimated in a vacuum, when lyophilization is complete, ampule neck sealed under vacuum.

Question 19

Methods of bacterial division

Answer 19

• binary fission (mainly)
• budding (plantomycetes, cyanobacteria, firmicutes)
• fragmentation (few filamentous bacteria)

Question 20

Lag Phase

Answer 20

Period of adjustment to new conditions where little to no division occurs (pop doesn’t increase); metabolic activity is high and this process may last 1hr-few days

Question 21

Log phase

Answer 21

generation time reaches a constant minimum, the number of cells produced greatly exceeds the number of cells dying, and the metabolic activity is at its highest; cells are most susceptible to adverse factors such as radiation or antibiotics.

Question 22

stationary phase

Answer 22

pop begins to stay static (cells producing = cells dying), cell number doesn’t increase, cell division slows down. Factors that can slow down microbial growth is the accumulation of toxins, acidic pH, limited nutrients or insufficient O2

Question 23

Death phase

Answer 23

population size decreases (cells dying > cells producing), cell number decreases logarithmically, cells lose ability to divide, and a few cells may remain alive for a long period of time.

Question 24

plate count

Answer 24

(DIRECT) spread plate with a sample and count number of colonies (assumptions are that each colony originates from a single cell and each inoculum is homogenous; the former isn’t always true). it measures only viable cells, but it takes 24hrs+, and only counts between 25-250 colonies are accurate. There are also plate counts, where you can add more culture or determine aerotolerance, but heat sensitivity could be a problem, embedded colonies may not be transferable, some differential plates require surface colonies for morphology diagnoses.

Question 25

Filtration

Answer 25

(DIRECT) used to measure dilute bacterial samples. first a sample is filtered and then the filter is transferred onto a petri dish to then incubate and then count the colonies.

Question 26

Most probable number

Answer 26

(DIRECT) used to measure bacteria that will not grow on solid medium. Make several dilutions of a culture and inoculate a broth tube for each one, then count the number of positive tubes in each set.

Question 27

Direct Microscopic count

Answer 27

bacteria/mL = # cells counted/ volume of area counted

(DIRECT) a bacterial susmension (0.01mL) is placed on a slide with a grid and the cells are counted and multiplied by a volume factor to obtain concentration. there is no incubation time required, but you cannot always distinguish between live and dead cells, motile bacteria are difficult to count, and it requires a high concentration of bacteria (10mil/mL).

Question 28

Turbidity

Answer 28

(INDIRECT) as bacteria multiply in a broth tube, the media becomes more turbid. there is no incubation time required, but you cannot distinguish between live and dead cells and it requires a high concentration of cells (10-100mil cells/mL)

%transmittance correlates to cells/mL; you plot the OD units on a graph against viable counts from plate counts (standard curve).

Absorbance/optical density = 2 - log % Transmittance

Question 29

Metabolic activity

Answer 29

(INDIRECT) assumes the amount of metabolic product is in direct proportion to cell number.

Question 30

Dry weight

Answer 30

(INDIRECT) best for measuring filamentous bacteria or fungi. cells are centrifuged to then be desiccated and weighed. Wet weight and volume of cells after centrifugation measurements can be made. However, it does’t distinguish live and dead cells.