Microbiology Lab Unit 2

What are the cardinal temperatures?

minimal, optimal and maximal temperatures at which a bacterium can grow

What are psychrophiles?

can only grow at temperatures less than 20 C

typical range -5 C to 20 C

80% of all bacteria can grow at 10 C or below

NOT HUMAN PATHOGENS

What are psychrotrophs?

typical range for growth 0 C to 30 C

CAN BE HUMAN PATHOGENS

found in soils, surface water and in foods

What are mesophiles?

optimal temperature for growth is near normal human body temperature

CAN BE HUMAN PATHOGENS

What are thermophiles?

can be found growing in hot springs

40-75 C

heat loving

What are hyperthermophiles/extreme thermophiles?

can come from thermal vents in ocean floor

65-110 C

What environment do acidophiles grow well in?

acidic environments

pH ~5 or lower

stomach acid

What environment do alkaliphiles grow well in?

basic/alkaline

pH ~8 or higher

alkali flats

What environment do neutrophiles grow well in?

neutral pH 5.5-8

MOST HUMAN PATHOGENS ARE NEUTROPHILES since bodily fluids are kept at a neutral pH

What is turgor pressure?

the pressure inside a cell that is required for survival

What could happen if the correct turgor pressure is not maintained?

1. lysis - cell rupture

2. plasmolysis - cell shrinkage

What is osmosis?

the movement of water from an area of low solute concentration to an area of high solute concentration in order to achieve equilibrium

What is osmotic pressure?

the force with which water flows from an area of low solute concentration to an area of high solute concentration

What is hypotonic?

HIGHER solute conc. INSIDE the cell than outside

low osmotic pressure

water moves INTO the cell

cell will lyse

What is isotonic?

equal solute conc. inside and outside of cell

water moves in and out of cell at equal rate

What is hypertonic?

LOWER solute conc. inside the cell than outside

high osmotic pressure

water moves OUT of cell

cell will shrink (plasmolysis)

What is salinity?

the concentration of salt in a solution

can affect osmotic pressure

What are halophiles?

salt loving bacteria

What are extreme halophiles?

a subgroup of halophiles that can ONLY survive at salt concentrations from 15-25%

What are osmotolerant bacteria?

bacteria that can survive and grow over a wide range of salt concentrations

What is aerotolerance?

the ability or inability to live in the presence of oxygen

What are obligate aerobes?

must have oxygen to survive and grow

What are facultative anaerobes?

can grow in the presence or absence of oxygen

What are aerotolerant anaerobes?

do not need oxygen but can survive in the presence of oxygen

What are microaerophiles?

survive ONLY when oxygen levels are low

What are capnophiles?

survive ONLY when CO2 levels are high

What are obligate anaerobes?

any oxygen will kill the bacteria

What are the agar deep stabs made of?

10 mL of enriched TSA which allows more types of bacteria to grow

Why is more media used at the bottom of an agar deep stab?

to ensure that the bottom of the tube is anaerobic

What is the result of an agar deep stab with no growth at the top of the stab line?

obligate anaerobe

What is the result of an agar deep stab with growth all along the stab line?

facultative anaerobe

What is the result of an agar deep stab with growth only at the very top of the stab line?

obligate aerobe

What are the 3 important ingredients in fluid thioglycollate medium?

1. sodium thioglycollate - reducing agent (reduces O2 to H2O

2. L-cystine - reducing agent (reduces O2 to H2O

3. resazurin - oxidation-reduction indicator - red/pink in the presence of oxygen. straw color in the absence of oxygen

What does growth look like for aerotolerant anaerobes in fluid thio. ?

sparse growth throughout tube

What does growth look like for facultative anaerobes in fluid thio. ?

thick growth near top of tube but also down to the bottom

What does growth look like for obligate anaerobes in fluid thio. ?

growth only at the bottom of the tube

no growth in the red colored oxygen containing portion of the media

What does growth look like for obligate aerobes in fluid thio. ?

no growth in the straw colored portion of the media that does not contain oxygen

only in the red portion (top of tube)

How is an anaerobic system created using a chemical gas generating packet?

gas packet contains inorganic carbonate, activated carbon, ascorbic acid, and water

packet becomes activated by exposure to air and CO2 gas is produced

anaerobic conditions achieved in 2.5 hours

How can the methylene blue indicator strip of the anaerobic jar be read?

blue in the presence of oxygen

white in the absence of oxygen

anaerobic conditions in the jar would show a white indicator strip

What are the 2 reasons why anaerobic conditions were not achieved with the anaerobic jar (strip stays blue)?

incomplete gasket seal

hole in pouch

What are the results of the anaerobic jar test for facultative anaerobes?

growth on aerobic and anaerobic plates

What does growth on only the aerobic plate mean? (AJ)

obligate aerobe

needs oxygen to survive

What does growth only on the anaerobic plate mean? (AJ)

obligate anaerobe

cannot survive in the presence of oxygen

T/F Is the majority of bacteria anaerobic?

True

can be found in soil, digestive tract, etc..

some anaerobic bacteria can be human pathogens but most are non-pathogenic

What is the plate count method and what are its pros and cons?

measures a bacterial population

pros: measures # of VIABLE cells (CFU/mL)

cons: time consuming and assumes EACH bacterium grows and divides to produce a SINGLE colony

What are the 4 direct methods of measuring the growth of bacteria?

1. plate counts
2. filtration and plate counts
3. most probable numbers (MPN)
4. direct counting

What is the filtration and plate count method and what are its pros and cons?

measures a bacterial population (CFU/mL) and is based on the fact that bacteria CANNOT pass through the pores of membrane filters - membrane then placed on agar plate and allowed to grow

pros: measures # of VIABLE cells in a small quantity of sample

cons: time consuming and assumes EACH bacterium grows and divides to produce a SINGLE colony

What is the most probable numbers method and what are its pros and cons?

statistical estimating method for measuring a population (MPN/100 mL)

pros: can be used for bacteria that CANNOT grow on solid media

cons: only a statement that there is a 95% statistical chance that a population falls within a certain range

What is the direct counting method and what are its pros and cons? (PH chamber)

measures the number of bacterial cells in a sample - manual counting of bacteria using a Petroff-Hausser counting chamber

pros: very accurate and inexpensive

cons: very tedious and time consuming

What is the direct counting method and what are its pros and cons? (C counter)

electronic cell counter (Coulter counter)

pros: very accurate and results obtained quickly

cons: expensive equipment that requires lots of training to operate

What are the 3 indirect methods of measuring bacterial growth?

1. metabolic activity
2. dry weight
3. turbidity

What is the metabolic activity method of bacterial growth?

estimates bacterial numbers by measuring the metabolic activity of a bacterial population

assumes that the amount of the metabolic product is directly proportional to the # of bacteria present

What is the dry weight method of bacterial growth?

estimates bacterial numbers by measuring the weight of the population

as population grows, greater number of cells, increases entire weight of population over time

What is the turbidity method of bacterial growth?

estimates bacterial numbers by measuring the turbidity of growing cultures in liquid medium

one of the quickest, easiest and most common methods

What is the relationship between turbidity and # of bacteria?

As turbidity decreases so does the number of bacteria - direct relationship

the more a culture is diluted, the less turbid it becomes

What is a spectrophotometer?

device used to measure the turbidity of a liquid sample

monochromatic beam of light (single wavelength) shone through a sample in order to measure the percent transmittance (%T) or the absorbance

What is transmittance?

fraction of incident light that passes through a sample

T=intensity of light OUT (I)/intensity of light IN (Io)

What is absorbance (optical density)?

quantity of light that a sample neither transmits nor reflects

related to % transmittance

direct/linear relationship between absorbance and concentration

At what absorbance value do we need to dilute the sample for the spectrophotometer?

1.99 A or above

sample diluted 1:5 ( 1 mL sample + 4 mL solution)
0.8x5=4

What is the relationship between absorbance and concentration?

linear relationship

low absorbance -> low # of bacteria

high absorbance -> high # of bacteria

What are the McFarland Standards?

a set of reference samples of different turbidity that can be used to estimate the number of bacterial cells in a sample

standards have specific bacterial cell concentration

Closed system growth formula?

Nf=Ni(2^n)

Nf = # of cells at final time point

Ni = # of cells at initial time point

n = # of generations

Can environmental factors have a direct impact on the doubling time of an organism?

yes

What is a closed growth system?

an environment in which NO nutrients are added and NO waste products removed

4 distinct phases of bacterial growth can be observed

Lag phase?

adjusting to new environment

NO cell division but time of HIGH metabolic activity preparing for growth

the more drastic the change in environment, the longer the lag phase

Log phase?

MAXIMUM growth rate, exponential

when plotted on log scale, straight line in growth curve

THIS IS WHEN GENERATION TIME OF A BACTERIUM IS DETERMINED

as long as sufficient nutrients are available and favorable conditions exist, exponential growth will happen

Stationary phase?

nutrients become limited, waste products accumulate, leading to unfavorable environment

growth rate = death rate -> no net change in cell numbers

length of this phase varies among species

Death phase?

all nutrients will become depleted and the levels of waste products will become toxic

bacterial begin to die

may be at a rapid rate or gradual

Why might the absorbance value change much during the death phase?

if bacteria do not lyse when they die, turbidity will probably not change and so a plate count should be done to see if death is actually occuring

if lysis happens, then absorbance decreases, easy to observe

What can a bacterial growth curve provide?

1. the length of time the bacterium is in each growth phase

2. the optimal growth conditions of a bacterium

3. the mean generation time of the organism

What did we determine in the closed system growth lab?

the growth of Vibrio natriegens in several salt concentrations to determine the generation time under each condition and to establish the bacterium's optimal growth environment

What does the OF test determine?

what catabolic pathway a bacterium can use to break down a carbohydrate

What are the 2 catabolic pathways of glucose?

aerobic respiration and fermentation

Aerobic respiration?

oxidation pathway

O2 final electron acceptor

oxygen environment required

Fermentation?

organic molecule final electron acceptor

occurs in the presence or absence of O2

Does aerobic respiration or fermentation produce large amounts of acidic compounds?

fermentation

AR produces a small amount

What are the 2 important media components in OF medium?

a carbohydrate

pH indicator bromothymol blue

What is the initial color of OF media?

What color does it turn when acidic?

green

yellow

Why is sterile mineral oil used?

to produce an anaerobic environment and promote fermentation

If organism can only use oxidation pathway? OF test

glucose can't be broken down in anaerobic conditions -> green

aerobic conditions -> moderate amount of acidic compounds made -> only media at top of tube turns yellow

If organism is capable of fermentation and oxidation? OF test

glucose broken down in both environments -> acidic compounds made -> both tubes yellow

If organism is weak or slow fermenter? OF test

glucose slowly broken down in both environments -> acidic compounds begin to accumulate -> both tubes yellow at top of tubes

If organism cannot metabolize the carbohydrate? OF test

glucose not broken down -> no acidic compounds made -> turns green or bluish green if alkaline products made (protein degradation)

Anaerobic respiration?

does not use oxygen as final electron acceptor

instead uses nonoxygen electron acceptor like nitrate (NO3-)

Denitrification?

nitrates into nitrogen gas

NO3- -> N2

Ammonification?

multistep process that leads to the production of ammonia from nitrate NO3-

What is the first step in both denitrification or ammonification?

the reduction of nitrate (NO3-) to nitrite (NO2) by the enzyme nitrate reductase

What is the purpose of the nitrate reductase test?

to determine if an organism can use nitrate for either denitrification or ammonification

What are the 2 key components in nitrate broth NR test?

potassium nitrate (KNO3) - the nitrate source

durham tube to monitor gas production

broth is clear because it has no color indicators

Do both denitrification and fermentation lead to the production of gases?

yes

Production of gas in a non-fermentor? NR test

gas must be produced by denitrification and must be nitrogen gas

organism must have nitrate reductase for denitrification

nitrate reductase POSITIVE -> test finished

Production of gas in a fermentor? NR test

type of gas produced not known -> test must be continued

Growth overnight but no gas production but organism IS a fermentor?

2 reagents added:
1. reagent A = sulfanilic acid
2. reagent B = naphthylamine

What does nitrite and water from the media make?

OH- and HNO2 nitrous acid

What does nitrous acid and reagent A (sulfanilic acid) make?

diazotized sulfanilic acid

What does diazotized sulfanilic acid and reagent B (naphthylamine) make?

p-sulfobenzene-azo-a-naphthylamine

RED COLOR

If a red color is present after the addition of reagents A and B, what does this mean?

means nitrous acid was available for reagents A and B to react with

nitrate reacted with water to produce nitrous acid

nitrate in broth converted to nitrite

SINCE NITRITE PRODUCED, ORGANISM IS NITRATE REDUCTASE POSITIVE

If no red color is present after the addition of reagents A and B, what does this mean?

NO nitrous acid available for reagents A and B to react with

NO nitrite available to react with water to produce nitrous acid

nitrate in broth but NO nitrite present

must continue with test

What are the 2 possibilities if no red color is seen after reagents A and B added?

1. organism does not have nitrate reductase and cannot convert nitrate to nitrite

2. organism has reduced all nitrate to nitrite and all nitrite has been converted to other nitrogen compounds

ADDITION OF ZINC POWDER REQUIRED

What does the addition of zinc powder do in NR test?

catalyzes the reduction of any nitrate source in the broth to nitrite