Microbio Exam 2

chemically defined culture media

know exact composition of pure chemicals (like MgSO_4 instead of tryptone)

complex/rich media

• we don’t know exact chemicals inside it

• used to grow organisms where we do not know their nutritional requirement

Nutrient broth is always complex. true or false?

true

selective media

only allows certain organisms to grow

BG agar

Yellow means e-coli

high growth/pink usually means salmonella

enriched media

organisms are picky & need more growth factors, vitamins, etc.

• usually add blood, eggs, serum

Differential media

contains certain compounds to help visually distinguish colonies (like pH)

• Alpha Hemo= RBC destroyed, partial clearning zone

• Beta Hemo= clear, colorless zone; RBC LYSED

• Gamma Hemo= no activity/discoloration

Eosin Methylene Blue (plate)

• Gram + cannot grow (Selective)

• Lactose in media; if broken down, green sheen is shown usually with e. coli

• purple/colorless means no lactose broken down

MacConkey Agar

• bile & crystal violet inhibit Gram +

• lactose fermenters make it pink

• good for clinical diagnostics on stool, urine, or wound cultures to isolate gram - bacteria

Mannitol Salt Agar (MSA)

• mannitol fermentation & phenol red → yellow

• salt selects for S. aureus

• good to isolate S. aureus

BSL 1

PPE, goggles, hand washing, autoclave, & surface decontamination

Culturing in Solid Media

• Stab/Deep cultures

• Slant cultures

• Plate cultures

Growing Aerobically

Step 1:

• CHNOPS

• Buffer

• Water

Step 2: Sterilize

Step 3: Inoculate

Batch Culture

• set volume & closed systems

• no nutrient renewal & causes exponential growth which is limited

Continuous Culture

• renews nutrients, removes waste products, and accumulates cells

• bacteria always grows at a constant density

BSL 2

Pathogens not transmitted by aerosol/contact

• same day decon, specialized training

BSL 3

pathogens transferred aerosol/ contact

• no public drain, protective lab clothing, hats, respirators, non circulating airflow, immunizations, and biosafety cabinet

BSL 4

readily transmitted pathogens by aerosol, contact and FATAL with no preventative vaccines/cure

• clothing change, shower, “space-suit”, no running water, & special exhaust system

What are biofilms good for?

Helps to share nutrients, disperse waste, & shelter from harmful factors

quorum sensing

bacteria are attracted to each other by…

Biofilm formation steps

1- attachment

2- growth

3- dispersal

Generation time

time it takes a bacterium to do 1 binary fission (2x)

Bacterial Growth

• occurs through binary fission, an asexual process

• short generation time

Binary Fission

(1.) Cell elongates & replicates DNA

(2.) Cell wall & plasma membrane constrict

(3.) Septum/crosswall forms

(4.) cells separate

LAG Phase

Length of time is variable

• dependent on how old inoculum is

• change in cell comp & limited cell division

• adaptation to new conditions

• resynthesis of damaged cells

LOG Phase

• Short

• # cell doubles in fixed time period

• cells are most active

• b/c gen time is a constant, a log plot can be drawn

• always use Log10 for plotting

Stationary Phase

• longest phase

• growth limited by nutrient availability, waste removal, & space

• Important time for 2 metabolic production (such as antibiotics) and endospore formation

Death Phase

• exponential loss of cells

#of generations

(Log # cells (final) — Log # cells (initial))/ 0.301

generation time

minutes/generation

Petroff-Hauser Cell Conter/ Direct Microscopic Count

• know volume of cells

• doesn’t distinguish live/dead cells

• difficult to see small/unstained cells

• labor intensive

• motile bacteria hard to count

CFU Calc

#colonies on plate x reciprocal dilution then convert to mL

Methods to Measure Growth

• Direct Microscopic Count

• Turbidity Measurement

• Viable Cell Count

Direct Microscopic Count

know volume and # cells/ml

• hard to distinguish living/nonliving cells

• labor intensive

• hard to count motile bacteria

• hard to see small unstained cells

Turbidity Measurement

• good for high count

• need spectrophotometer

• dead cells are counted

• super fast & doesn’t destroy cells

Viable Cell Count

• large # of bacteria

• sensitive

• great for living cells

• use serial dilutions

• assumes cell microbes grow on the media

• need 30-300 colonies

Limitations of Viable Cell Count

• pipetting error

• diluting error

• viability

• poor mixing

• clumps/chains cells develop into 1 colony

Disinfection

use of physical/chemical agent to kill microbes

antiseptics

• chemicals applied to living tissue

disinfectants

• chemicals applied to inanimate objects

Aseptic technique

• techniques to prevent microbial contamination in lab, other ppl, instruments, medicine, food & yourself

Sterilization

• death of all cells (whether they are dormant or vegetative)

Bacteriostatic

• agents inhibit growth, but don’t kill microbes

• often reversible

Bacteriocidal

agents that kill bacteria

• inhibits protein synthesis & loss of DNA sensitive

Bacteriolytic

• agents lyse cells & release cytoplasm

• irreversible

Bacteria die at a constant/variable rate

constant

Death Curve

D= decimal reduction time

What is the death curve affected by?

impacted by population load, env factors, and time

longer treatment = more effective

What happens with Heat with the Death Curve?

higher temp compensates for longer exposure

Physical means of microbe removal

Heat

Radiation

Filtration

Food Irradiation

• uses gamma, x-rays, & e- beams

• destroys insects, pathogens, inc shelf-life, sprout inhibition, & quarantine

Moist Heat

• boiling (does not rid of endospores)

• pasteurization (not sterilization)

  • reduces spoilage & pathogens, although some can survive

autoclave

• steam under pressure

• high temp above boiling & 2x the pressure (15psi)

• denatures proteins

• DOES STERILIZE

Phenols & Phenolics (Lister)

• disrupt plasma membranes, inactivate enzymes, and denature proteins

• good for disinfection of surfaces & instruments

Biphenols

• disrupt plasma membrane & stop fatty acid synthesis

• often used as disinfectant and in prescription lotions

alcohols

• denature proteins, dissolve lipids

• requires water

• bacteriocidal

• antiseptic

• disinfectant

Tinctures

iodine dissolved in alcohol

Iodophors

iodine dissolved in organic molecules

• alter protein synthesis & membranes

• for skin

HCL

• oxidizing agent

Biguanide compounds

• bacteriostatic and bacteriocidal

• used as both an antiseptic and disinfectant

• destroys cell membrane

• not effective against pseudomonads, endospores, and some viruses

Acid-anionic detergents

• sanitizing

• damages cell membrane & enzymes

Quartenary compounds

• damages plasma membrane & denatures proteins

• bactericidal

Heavy metals

• biocidal and antiseptic

• ions combine w/ sulfhydryl groups on proteins & denature them

Disk-Diff method

evaluates efficacy of a chemical agent

• if zone of inhibition present, that means it is working

Chemoauto vs chemolitho

chemoauto uses organic cmpnds while chemolitho uses inorganic cmpnds (H_2, etc)

oxidative is catabolic. True or false?

true

reductive is catabolic / anabolic

anabolic

3 ways to generate ATP

• substrate level phosphorylation

• oxidative phosphorylation

• photophosphorylation

substrate level phosphorylation

high energy phosphate containing molecule transfers Phosphate to ADP in enzyme mediated chemical reactions to make ATP

Oxidative phosphorylation

through a series of electron transfers (redox) to make energy to drive ATP production (ETC)

Which cycles are present in bacteria—glycolysis or krebs?

trick question! BOTH

Which stage occurs in both cellular respiration & fermentation?

glycolysis

goal of glycolysis

glucose → pyruvic acid through oxidation which makes ATP & NADH.

• can occur w/o oxygen

Prep Stage glycolysis

2 ATP used to split glucose into DHAP & GP (MORE GP PRODUCED & REVERSIBLE)

energy conserving stage of glycolysis

2 molecules oxidized to 2 pyruvic acid

• produces 4 ATP & 2 NADH

• substrate-level

Goal of krebs cycle

• oxidation of acetyl-CoA produces some ATP, but main is to reduce coenzymes NADH & FADH.

• breaks down complex molecules (catabolic)

bridge/transition step

pyruvic acid is oxidized & decarboxylated

• NAD+ reduced to NADH

• CO2 released

What’s the bond connecting the acetyl group & conenzyme A group called?

thioester bond

• super unstable & exergonic

After 2 cycles for 2 pyruvic acids, what is produced?

• 2 ATP (substrate-level)

• 8 NADH

• 2 FADH2

1 NADH=3 ATPS

1 FADH2= 2 ATPS

what is the total amount of ATP you get if you only did glycolysis and krebs cycle (for prokarya)?

38 ATP

how do electrons transfer through electron carriers?

concurrent proton translocation

chemiosmosis

• electrochemical gradient to generate ATP

• uses potential energy to drive ADP → ATP

oxidative phosphorylation

ATPase-catalyzed ATP production if proton motive force originates from cellular respiration rxn

where do bacteria have ETC?

in plasma membrane

Heme core

has metal ion, can be reduced/oxidized

reduction potential (how good of an e- acceptor you are)

negative is best

oxygen is final e- acceptor in aerobic respiration

Fermentation

• can be aerobic/anaerobic

• just substrate level phosphorylation

• final e- acceptor is a organic molecule

Nitrate reduction

NO3- TO NO2-

denitrification

NO3 to NO, N2O, or N2

alcohol fermentation

produces ethanol & CO2

lactic acid fermentation

produces lactic acid

homolactic acid

produces lactic acid only

heterolactic acid

produces lactic acid and other compounds

HOPS plant is bacterio___

cidal

in lactic acid fermentation, what is the final e- acceptor?

pyruvate

bacteria first appeared…

3.9 BYA

16S rRNA

• present in all bacterial cells

• small subunit ribosome

• has both variable and constant regions, so you can compare what is conserved and what isn’t

• hard to “remove”

• horizontal transfer DOES NOT OCCUR

Genetic expression

replication, transcription, and translation

vertical gene transfer

parent → daughter cells

same DNA for everyone

horizontal gene transfer

DNA given to a cell of the same generation

• bacteria unique of this