Microbiology Unit 2

Front 1

Energy source and essential for cellular structures and functions

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carbon

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2 ways microbes obtain carbon

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autotrophs and heterotrophs

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able to take up atmospheric CO2 usually using light to make sugars

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autotrophs

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degrade organic carbon molecules found in the medium (primarily glucose)

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heterotrophs

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necessary for macromolecules including proteins DNA and RNA

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nitrogen

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sources of nitrogen

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1.) atmospheric N2 (ex. rhizobium)
2) inorganic compounds such as salts of ammonium (NH4) and nitrate (NO3)
3) organic compounds such as amino acids

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Other non- metallic elements needed for microorganisms, what they are used for, and where they come from

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1) phosphorus- used in nucleic acids (RNA, DNA). Comes from salts of phosphate (PO4)
2) Sulfur- needed for some amino acids. Sulfur sources include elemental sulfur and sulfates (SO4)

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Trace amounts of zinc, sodium, potassium, copper, manganese, magnesium, and iron as well as other metals are used in _______

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osmosregulation, electron transport and enzyme regulation

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trace amounts needed for cellular growth and activities

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vitamins

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necessary for nutrient uptake

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water

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What are the types of intracellular energy and define them

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1.) Phototrophs- radiant energy
2.) Chemotrophs- chemical energy

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Types of physical variables of microbial growth.

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Temperature, ph, atmosphere,

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Temperature

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affects rate at which biochemical reactions occur

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Optimal growth temp

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20-40 C

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What happens at too low of temps?

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slowed enzyme activity

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what happens at too high temps

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enzymes can denature and wont function

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measure of H concentration

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pH

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Too low or too high a pH can cause

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harm to enzymes and thus slow growth

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best pH for growth

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Neutral 7

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some bacteria require O2 while others will not grow in the presence of O2

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atmosphere

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made from known amounts of pure chemical compounds

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chemically defined media

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advantages of a chemically defined media

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know exact composition

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disadvantages of a chemically defined media

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not routinely used because must know the exact nutritional requirements of the microrganism

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commonly used and works to grow most bacteria. Exact chemical composition is not known

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artificial/ complex media

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contents of a nutrient broth

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basic medium composed of peptone (nitrogen source), and beef extract (adds organic carbon, nitrogen, salts, and vitamins)

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contents of yeast extract broth

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an enriched medium composed of basic artificial medium with yeast extract, vitamins, or blood to support growth of fastidious microbes with very specific nutritional needs.

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a medium that distinguishes between multiple bacteria by appearance of colonies grown on a plate. For example, colonies of various bacteria may be colored differently

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differential medium

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a medium that suppresses growth of one type of bacteria, but permits the growth of other kinds of bacteria.

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selective medium

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Mannitol salt: differential? How? Selective? How?

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differential by mannitol fermentation, selective by only cells that can grow on 7.5% NaCl survive. EX) stapyloccocus sp.

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blood agar (enriched medium): differential? How? Selective? How?

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differential by hemolysis of red blood cells, not selective

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MacConkey: differential? How? Selective? How?

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differential by lactose fermentation as well as E. coli vs other fermenters, selective by only gram negative grow

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Eosin methylene blue (EMB): differential? How? Selective? How?

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differential by lactose fermentation as well as E. coli vs other fermenters, selective by only gram negative grow

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Phenlethyl alcohol: differential? How? Selective? How?

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not differential selective by only gram positive grow

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lowest temperature at which growth occurs

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minimum growth temperature

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highest temp at which growth occurs

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maximum growth temperature

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temperature at which reproductive rate is greatest

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optimum growth temp

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Enzyme activities _______ with temperature

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increase

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the temperature at which the enzymes are denatured

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70 c

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the temperature is lowered toward freezing, enzyme inactivation occurs at what point

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0 c

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(-5 to 20 c) grow at temperatures from 0-5 c

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psychrophiles

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(20-45 c) grow at 37 c but cant grow above 45 c

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mesophiles

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optimum growth temperature of 20-30 c occurs o what

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plant saprophytes

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mesophiles w/ optimal growth temperatures between 35-40 c

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mesophiles that grow on warm blooded hosts

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will grow at 37 c however optimal growth is between 45-60 c

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facultative thermophiles

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grow only at temps above 50 and optimal growth temps are greater that 60 c

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obligate thermophiles

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measure of acidity

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pH

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14 on pH scale

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basic

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1 on pH scale

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acidic

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optimal pH of bacteria

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6.5-7.5

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optimal pH of fungi including yeasts

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4-6 (grow i more acidic conditions)

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substance used to prevent a change in the pH of a medium

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buffer

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produced during carbohydrate degradation

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acids

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produced during protein degradation

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bases

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any biochemical process in which energy is liberated

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respiration

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4 characteristics of aerobes and 2 examples.

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1.) require O2 for growth
2.) O2 final electron acceptor
3.) possess catalase (H2O2---- > H2O+ O2)
4.) grow near surface of media
ex) bacillus, neisseria

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4 characteristic of anerobes and 3 examples

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1.) O2 absence
2.) intermediate organic electron acceptor
3.) strict anaerobes are killed by O2. Lack catalase and superoxide dismutase
4.) grow at bottom of media
ex) clostridium, peptococcus, peptostreptococcus

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3 characteristics of facultative anerobes and examples

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1) can grow if O2 present or absent
2) complete oxidation of glucose in O2
3.) incomplete oxidation w/out O2
examples) e. coli and most enteric bacteria

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3 characteristics and 1 example of microaerophillic

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1.) grow under reduced oxygen tension
2.) large amounts of O2 block oxidative enzymes
3) grow just below surface of medium
ex.) saccharomyces cerevisiae

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lowers oxidation reduction potential of medium (combines w/ O2)

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sodium thioglycollate

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contains glucose and resazurin a redox potential indicator

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fluid thioglycollate medium

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contents of a gaspak jar and what they do

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1.) indicator- filter paper + methylene blue. blue-oxidized o2 present. Colorless- reduced, 02 absent
2.) foil envelope- sodium borohydride tablet, sodium bicarbonate tablet, citric acid tablet, and palladium catalyst

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two types of metabolic reactions

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1) catabolic
2) anabolic

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reactions that breakdown or degrade complex organic molecules

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catabolic

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reactions that use energy to assemble small molecules into larger molecules

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anabolic

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the sum total of all metabolic reactions w/in the cell

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cellular metabolism

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bacteria produce 2 general types of enzymes

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extracellular and intracellular

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secreted outside the cell

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extracellular enzymes or exoenzymes

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mainly hydrolytic and used to degrade larger molecules such as polysaccharides, lipids, and proteins into smaller molecules. Tests used to determine production of extracellular enzymes include

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starch, casein, lipid, and gelatin hydrolysis

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responsible for synthesis of new molecules and energy production (fermentation). waste products from these reactions are excreted into the medium

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intracellular enzymes or endoenzymes (within the cell)

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tests to determine production of intracellular enzymes

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carbohydrate fermentation, hydrogen sulfide production, and triple sugar-iron (tsi) test

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a glucose polymer linked together by glycosidic bonds

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starch

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test for starch hydrolysis

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starch agar (NA+ salt) is used. Grams iodine is added to the area of growth. Starch in the presence of iodine yields a blue-black color (negative). Hydrolyzed starch produces a clear zone (positive)

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a milk protein composed of amino acids linked together by peptide bonds

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casein

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Before assimilation into the cell, proteins must be broken down step by step to their basic units, amino acids. Breakdown occurs thru the process of

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proteolysis or peptonization

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test for protein hydrolysis

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organisms grown on casein agar (NA+milk) will produce a clear zone when hydrolysis has occurred (positive) or remains opaque w/out hydrolysis (negative)

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a protein produced by hydrolysis of collagen. It exists as a liquid above 25 c and a gel below 25 c

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gelatin

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gelatin test

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cultures that remained when chilled after incubation are examples of organisms that produce gelatinase. Gelatin hydrolysis can be rapid or slow

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Types of extracellular enzymatic activities

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starch hydrolysis, lipid hydrolysis, protein hydrolysis and gelatin hydrolysis

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organisms gain energy from utilization or bioxidation of carbohydrates

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biooxidative pathways

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bacteria use 2 ways to break down carbohydrates

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1) fermentation
2) respiration

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Glycolysis is the relevant pathway with pyuvic acid the main intermmediate compound. Bacteria vary with respect to what happens to the pyruvic acid. Different species have different organic acids (lactic, formic, or acetic) at the end

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fermentation

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organic acid endproducts of fermentation will lower the ______ of the medium

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pH

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Use ________ to detect changes in pH for fermentation

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phenol red
pH 6.8 or less-yellow
pH 7 or above- red

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biooxidation in which O2 can serve as a final electron acceptor

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aerobic respiration

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biooxidation in which inorganic ions other than O2 (ie NO3- or SO4-) can serve as final electron acceptors

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anaerobic respiration

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test is used to determine ability of oranisms to ferment (degrade) a specific carbohydrate incorporated into a basal medium. Look for acid production as well as acid w/ visible gas production

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carbohydrate fermentation

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glucose fermentors:

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all members of the enterobacteriaceae

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glucose and lactose fermentors

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e. coli, klebsiella and enterobacter

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test media for carbohydrate broths include

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Nutrient broth + carbohydrate + phenol red
pH 6.8 yellow, pH 7.0 red

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used to check for gas production in carbohydrate broths

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durham tubes

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uninoculated carbohydrate broth

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reddish orange

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purpose of test is to differentiate among enterobacteriaceae which are all gram negative bacilli capable of fermenting glucose

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Triple sugar-iron test (TSI)

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enterobacteriaceae can all ferment _______

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glucose

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TSI Media contnts

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Phenol red + .1% glucose + 1% lactose and 1% sucrose, peptones, sodium thiosulfate, and ferrous sulfates

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method to streak tsi slant

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stab and fishtail

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acid production occurs with ______

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fermentation

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carbohydrate broth turns yellow

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positive (fermentation/acid production

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carbohydrate broth turns yellow with air bubbles

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(fermentation/ acid w/ gas production), positive w/ gas

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orange- red color, unchanged medium

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no fermentation, no acid production, Negative

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red slant/ yellow butt

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K/A, glucose fermentation, alkaline/acid

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Yellow slant/ yellow butt

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A/A, acid over acid, glucose/ lactose/ sucrose fermentation

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cracks or fissures in medium

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gas production

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black precipitate

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sulfur production

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only works for gram negative organisms otherwise it would make a false positive most likely A/K

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TSI slant

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Alkaline/no change

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K/NC, unable to ferment glucose

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IMViC stands for what

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Indole, methyl red, boges-proskauer, citrate

Front 107

Tryptophan, an essential amino acid, can be oxidized by some organisms

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Indole test

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Indole test

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SIM agar which contains tryptophan. Kovacs reagent (P- dimethylaminobenzaldehyde) is added to growth. Organisms that produce indole yield a red layer at the topd of the tube (positive rxn)

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qualitative test of acid produced from oxidation of glucose

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methyl red test

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e.coli produces _______amounts of acid

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large

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e. aerogenes produces ____________ acidic products

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neutral or non acidic products

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Methyl red test

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organism is grown in MR_VP broth. Methyl red indicator is added to culture to identify amount of acid present. When organism is e.coli medium pH=4 and is red. When acid is present but as much lower concentration pH=6 the medium turns yellow

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determines the ability of an organism to produce non-acid or neutral end products from organic acids present following glucose metabolism

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Voges-Proskauer test

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Voges- Proskauer test

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organism is grown in MR-VP broth. Bbarritt's reagent is added to culture. Pink/red color indicates presence of acetylmethycarbinol (positive reaction)

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some organisms use citratee as a carbon source for energy when glucose or lactose is not present. These organisms have 2 primary enzymes

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citrate permease and citrase

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citrate test

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Simmon's agar slants are used. Bromthymol blue indicator is incorporated in the medium. If culture grows (citrate positive, pH 7.6) media turns blue. IF no growth, medium is green (pH 6.9) and culture is citrate negative.

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Used to differentiate gram-negative enteric bacteria

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IMViC test results

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SIM test

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Ceck for black sulfide, look for branching from main stab, add Kovacs reagent (p-dimethylaminobenzaldehyde to tube. Red layer on top produces indole, no change negative

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to identify glucose oxidation with acid production. USed primarily to differentiate E. coli from E. aerogenes. Transfer 1/3 of culture to sterile tube to be used in V.P test. Add a few drops of methyl red to original culture

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Methyl red test. Positive red medium (PH=4, acid present, organism is E. coli).
Negative- yellow medium (pH=6, acid present but at low concentration)

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add 10 drops of barritts reagent A and shake. Add 10 drops of barritts reagent B and shake. Check culture every 3-4 minutes for 15 minutes

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voges-proskauer test

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positive test for voges-proskauer test

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rosy pink/ red medium (presence of acetylmethylcarbinol

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negative test for voges-proskauer test

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no change in medium (absence of acetylmethylcarbinol

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Colorless H2S gas combines with _________ to form insoluble black precipitate

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iron salt (ferrous ammonium sulfate)

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hydrogen sulfate test

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Sim media which contains sulfur substrates (peptone, sodium thiosulfate), ferrous ammonium sulfate and agar to make the medium semi-solid. A single stab inocculation is used. Insoluble black precipitate forms rxn is positive

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a number of organisms (proteus, klebbsiella, morganella, providencia) produce the enzyme urease which __________-

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hydrolyzes urea to CO2, water and ammonia

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urease test :

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Urea agar which contains urea, phenol red, buffers and yeast extract. Heavy inocculum is required for this test. Urea hydrolysis (ammonium carbonate production) causes an alkaline environment and indicator reflects pH=8.4 and turns deep pink/red (positive reaction). If medium is unchanged (pH 6.8) no urease production (negative)

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anaerobic condition where bacteria obtain oxygen from NO3 to use as an electron accepter. Nitrate reductase

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Nitrate reduction test

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medium for nitrate reduction test

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NB + .1% KNO3

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after inoculation and incubation, add reagents __________ and ________ which react w/ NO2.

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sufanilic acid and a- naphthylamine

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2 possibilities of a negative result of nitrate reduction test after 1st reagent added

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nitrate wasn't reduced or nitrate was reduced to nitrogen gas

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with no change on the nitrate reduction test what can you add to try to reduce nitrates to nitrites

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zinc which if zinc turns the medium red the test is negative because it means nitrate was not reduced at all by the 1st reagent

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enzyme used by aerobic organisms to catalyze oxidation of reduced cytochrome by oxygen

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cytochrome oxidase

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most gram positive organisms are oxidase __________

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negative

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2 bacteria that are oxidase positive

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pseudomonas and alcaligenes

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reagent used in oxidase test

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dimethyl-p-penylenediamine hydrochloride

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an enzyme used to break down hydrogen peroxide which is formed during aerobic respiration

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catalase

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used to distinguish streptococcus (negative test) from staphloccocus and micrococcus (positive test)

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catalase test

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bubbles in hydrogen peroxide

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catalase positive test