Exam 2

summary of decarboxylase test

• name of medium: decarboxylase broth

• substrate: lysine/ornithine

• bacterial enzyme: LDC/ODC

• end products: cadaverine/putrescine

• positive result: purple/alkaline

• negative result: yellow/acidic

• reagent: n/a

• pH indicator: bromocresol purple

summary of phenylamine deaminase

• name of medium: phenol alanine slant

• substrate: phenylamine

• bacterial enzyme: phenylamine deaminase

• end products: phenyl pyruvic

• positive result: green

• negative result: yellow

• reagent: Ferric chloride

• pH indicator: n/a

summary of casein hydrolysis

• name of medium: skim milk agar

• substrate: casein

• bacterial enzyme: casease (exoenzyme)

• end products: amino acids and polypeptides

• positive result: milk becomes clear

• negative result: no clearing, but still growing

• reagent: n/a

• pH indicator: n/a

summary of urease test

• name of medium: urease broth

• substrate: urea

• bacterial enzyme: urease (exoenzyme)

• end products: ammonia and carbon dioxide

• positive result: pink

• negative result: yellow

• reagent: n/a

• pH indicator: phenol red

summary of the triple sugar iron test (sulfur reduction)

• name of medium: triple sugar agar

• substrate: cysteine and sodium thio sulfate

• bacterial enzyme: cysteine desulfurize and thiosulfate reductase (various endoenzymes)

• end products: h2S + ferrous ammonium sulfate → black

• positive result: black for sulfur reduction

• negative result: not black

• reagent: ferrous ammonium sulfate

• pH indicator: n/a

summary of triple sugar iron (sugar)

• name of medium: triple sugar agar

• substrate: glucose/ lactose/ sucrose

• bacterial enzyme: various endoenzymes

• end products: acids and gas

• positive result: yellow

• negative result: pink/orange

• reagent: n/a

• pH indicator: phenol red

purpose of decarboxylase test

to determine whether bacteria can decarboxylate lysine or ornithine (amino acid)

peptones, beef extract in decarboxylase test

food for general growth

glucose in decarboxylase test

for fermentation ( bacteria use glucose 1st at a low concentration to lower pH)

pyridoxyl in decarboxylase test

vitamin B6 - required coenzyme

l-lysine or l-ornithine in carboxylase test

amino acid substrate

negative result meaning in decarboxylase

bacteria metabolized used a produced acid sugar that lowered the pH

purpose of deaminase

to determine whether bacteria can deaminate phenylaline

yeast extract in phenylalanine deaminase

general growth

Aspartame (nutrasweet & equal)

artificial sweetener that is a dipeptide of phenylamine and aspartic acid with methanol, 180x times sweeter than sugar

Why can’t you use a pH indicator as a readout

ammonia is a base and phenyl pyruvic acid is an acidic → cancel each other out

negative result of deaminase

stays yellow and no phenylpyruvic acid produced

positive result of deaminase

turn green and phenylpyruvic acid present

aerobic in deaminase

proteins are deanimated

anaerobic in deaminase

proteins are carboxylase

purpose of casein hydrolysis

to determine whether bacteria can hydrolyze casein

pancreatic digest of casein and yeast extract in casein hydrolysis

general growth

non-fat milk in casein hydrolysis

source of casein (substrate)

glucose in casein hydrolysis

added nutrients for growth

why peptones instead of intact proteins in casein hydrolysis

peptones are small and already digested. They’re ready to go for bacteria in terms of general growth

one type of organism in urease test

Proteus mirabilis

purpose of urease test

to determine whether bacteria can hydrolyze urea

yeast extract in urease test

general growth

urea in urease test

substrate found in urine

phenol red in urease test

pH indicator

purpose of TSI agar

to differentiate bacteria on the basis of glucose fermentation, lactose fermentation, sucrose fermentation, and sulfur reduction

peptones in TSI agar

general growth

glucose in TSI agar

low glucose concentration as substrate for fermentation to produce acids

lactose and sucrose in TSI agar

substrate (disaccharide) for fermentation to produce acid

ferrous ammonium sulfate in TSI agar

H2S detector reagent - turns black

sodium thiosulfate in TSI agar

source of reducible sulfur

phenol red in TSI agar

phenyl red

interpretation and organism of pink slant and yellow butt

• glucose only fermenter

• as glucose is exhausted, the organism will break down protein and cause reversion in the aerobic slant

• proteus Alcofacies

interpretation and organism of yellow slant and yellow with gas butt

• ferments glucose and lactose and/or glucose

• Eserichia coli

interpretation and organism of pink slant and orange butt

• non-fermenter, used peptones aerobically

• obligate aerobe (peptone used aerobically only)

• n/a

interpretation and organism of pink slant and pink butt

• nonfermented, used peptones anaerobically too

• non-saccharolytic

• pseundoneas aeroginosa & alicligenes faecalis

interpretation and organism of yellow slant and black butt

• H2S production and all sugar fermentation

• hydrolyzes into H2S by acidic

• pseudomonas vulgaris

interpretation and organism of pink slant and black butt

• H2S production and glucose only fermenter

• Proteus mirabilis & salmonella underaesis

purpose of motility test

to determine whether bacteria are motile (can move)

purpose of gelatin hydrolysis test

to determine whether bacteria can hydrolyze gelatin

gelatin

protein extract from bones, cartilage, connective tissue, etc.

• collagen is the most abundant protein in the body

• it is a protein food source for bacteria

organism in gelatinase hydrolysis test (+ bacteria)

Clostridium perfringens

• has collagenase (a type of gelatinase) activity

• causes gangrene: skin becomes black and dies. Blood can’t get to it so it can’t be treated with antibiotics

Pseudomonas aeruginosa

• most common cause of death in burn victims

• gram negative rod that loves water and if it gets into wound and get trapped in the dressing, almost no antibiotics work

purpose of anaerobic jar

to determine the oxygen requirements of an organism

O2 indicator and interpretations

• resazurin (O2 indicator)

• pink = oxidized/aerobic

• clear = reduced/anaerobic

how microbes process oxygen

Aerobes and the different types

• use molecular O2 as a final electron acceptor

• obligate aerobe

• microaerophiles

obligate aerobes and example

• require O2

• Pseudomonas aeruginosa

• superoxide dismutase = +

• catalase = +

microaerophiles and examples

• require low O2 concentration

• helicobacter pylori

• superoxide dismutase = - (a little)

• catalase = - (a little)

anaerobes and types

• don’t use O2 or it is toxic to them

• obligate anaerobes

• facultative anaerobes

• aerotolerant anaerobes

obligate anaerobes and example

• cease growth or die in O2

• clostridium

• superoxide dismutase = -

• catalase = -

facultative anaerobes and example

• grow with or without O2, but grow best with O2

• eserichia coli

• superoxide dismutase = +

• catalase = +

aerotolerant anaerobes and example

• can grow in O2, but don’t use it as final electron acceptor

• streptococcus

• superoxide dismutase = +

• catalase = -

purpose of thioglycollate medium

to determine the O2 requirements of an organism

yeast extract, peptones in thioglycolate medium

general growth

glucose/dextrose in thioglycolate medium

fermentation

sodium thioglycolate and L-cystine in thioglycolate medium

reduce O2 into water (make anaerobic

resazurin in thioglycolate medium

O2 indicator

• pink = oxidized, O2 present

• colorless = reduced, O2 absent

pseudomonas aeruginosa (gram - rod) in anaerobic jar

• aerobic = +

• anaerobic = -

• O2 requirement = strict/obligate aerobic

• disease = skin infection in burn victim, ear infection, lung infection in cystic fibrosis patients

• additional notes = pyocyanin pigment; interfaces with air

staphylococcus aureus (gram + coccus) in anaerobic jar

• aerobic = +

• anaerobic = +

• O2 requirement = facultative oxygen

• disease = skin infection, skin boil, abscesses, sepsis, food poisoning, pneumonia

• additional notes = staphyloxanthin pigment; no flagella

clostridium sporogenes (gram + rod)

• aerobic = -

• anaerobic = +

• O2 requirement = strict/obligate anaerobe

• disease = other clostridium, botulism, tetanus, gangrene, colitis

• additional notes = nitrate reducer, doesn’t interface with air; has flagella

neisseria meningitidis or N. gonorrhoeae (Gram - coccus) in anaerobic jar

• aerobic = -

• anaerobic = -

• O2 requirement = microaerophile (little air lover), requires low O2

• disease = meningitis, gonorrhea

purpose of oxidase test

to test bacteria for the oxidase enzyme

positive and negative result of oxidase

• positive = blue/purple; using O2 as FEA

• negative = colorless; does not use O2 as FEA (within 20 seconds)

purpose of catalase test

to test bacteria for the catalase enzyme (produced by organisms that use O2 as FEA in the ETC → neutralize hydrogen peroxide (H2O2))

positive and negative result of catalase test

• positive result = bubbles when H2O2 is added (O2 produced

• negative result = no bubbles when H2O2 is added (no O2 produced)

summary of gelatin hydrolysis

• name of medium: gelatin

• substrate: gelatin

• Bacterial enzyme: gelatinase (exoenzyme)

• End products: polypeptides and amino acids

• positive result: blue

• negative result: green

• reagent: n/a

• pH indicator: n/a

liquification

turning to liquid

purpose of nitrate reduction test

to determine whether bacteria can use nitrate (NO3) as the final electron acceptor (FEA) during anaerobic respiration

ammonification

the production of ammonium

denitrification

loss of biologically available nitrogen; occurs by release of nitrogenous gases

oxidation

loss of e (H) or gain of O2

reduction

gain of e (H) or less of O2

nitrate reduction substrate

NO3 (nitrate)

bacterial enzyme in nitrate reduction test

Nitrate reductase (endoenzyme)

product of nitrate reduction test

NO2 (nitrite)

complete process of nitrate reduction test

Color results in nitrate reduction test

zinc in nitrate reduction test

• a reducing agent, will cause nitrate to gain electrons

• turns red = bacteria don’t use nitrate

• remains clear = full reduction of nitrate occurred

reagent in nitrate reduction test

reagent A and B

Picture of pH effect on Lactobacillus

• acid = more murky

• base = more clear

pH effect of S Aureus

mostly murky around neutral

pH effect of A faecalis

• acid = more clear

• base = more murky

effect of staphylococcus epidermidis in osmotic pressure

grew at all concentrations of salt (from 2-11%)

effect of Escherichia coli in osmotic pressure

can only grow at certain salt concentrations; lives in gut and can’t tolerate changes in salt

osmotolerant

lives on skin and salt concentration can change

Chart of effect of temperature on microbial growth

temperature microbial growth chart

temperature effect on P. fluorescens

most growth around 5 C (psychotroph)

purpose of steam serilization

to determine if the autoclave is working properly to sterilize instruments, media, etc.

sterilization

killing all living organisms

sanition

reduce amount of micorobes

bacillus stearothermophilus endospores in steam steralization

heat resistant

bromocresol purple in steam sterilization

pH indicator

• glucose = acidic

• purple = alkaline

glucose in steam sterilization

fermentation if organisms survive, but will ferment glucose and produce acidic products; decrease pH

purpose of kirby bauer method

to perform a culture and antibiotic sensitivity test on an organism