Micro lab exam 2

If an organism isn't making ATP by cellular respiration, by what mechanism is ATP being generated?

Fermentation

Can Group A&B Streptococci be differentiated from each other by observing the results of their Gram stains and hemolytic reactions on blood agar? If not, what test can differentiate between them?

No, both are gram + and beta hemolytic. They can be differentiated by the bacitracin test.

Nitrification:

an anaerobic process and a reduction reaction; the charge is going down

Triple Sugar Iron Agar

differential medium that distinguishes members of Enterobacteriaceae by differences in their ability to ferment glucose, lactose, and sucrose, produce gas while fermenting them, and produce hydrogen sulfide (H2S). Color indicators: 1) phenol red (pink at pH 7.4 and higher, yellow at pH below 6.8, and orange between this range) 2) ferrous ammonium sulfate which turns black in presence of hydrogen sulfide. A false- may be observed if test is read after 24 hours.

anaerobic cellular respiration

final electron acceptor is an inorganic molecule such as nitrate, nitrite, or sulfate

Identify and briefly describe all bacterial cell structures that allow bacteria to move independently:

flagella, pili, endoflaeglla

If you removes the ferrous sulfate from the tube, what would you no longer be able to detect?

hydrogen sulfate

Lactose nonfermenters in Macconkey agar:

hydrolyze the peptones, ammonia builds up as a waste product raising the pH in the medium. Salmonella, Shigella.

Reversion

if after a few days the phenol red broth turns bright pink, giving a false negative.

The nitrate test differentiates bacteria on their ability to produce the enzyme:

nitrate reductase

deamination (phenol red test)

non-fermenting organisms are capable of doing this, possessing enzymes that can hydrolyze the peptone amino acids, releasing ammonia (NH3). Causes the pH to rise above 8.2 and the color becomes hot pink

Lancefield grouping:

separated into groups A-T based on specific carbohydrate composition of antigens on the bacterial cell surface. Three medically important groups: A- Streptococcus pyogenes, B- Streptococcus agalactiae, and D- streptococci and enterococci. Group N- Lactococcus lactis, important in production of buttermilk and cheese. Viridans group- streptococci.

pyruvic acid

the organic molecule that acts as the electron acceptor in fermentation

If organism is catalase-,

will not grow on MSA (exception for E. faecalis).

#cells/ml:

# of CFU's/(DF-final) X (volume plated)

Regarding bacterial families, what does an oxidase negative test result indicate? What does oxidase positive test result indicate?

-: Enterobacteriacea +: Neisseria

Equation for catalase reaction:

H2O2 > H2O + O2. H2O2 is the substrate, catalase is the enzyme.

List all 5 characteristics of Enterobacteriacea:

-all small gram- bacilli and some are coccobacilli -oxidase negative -facultative anaerobes -all saccharolytic, capable of fermenting glucose into lactic acid or other acidic compounds. -most capable of reducing nitrate to nitrite

TSIA results:

-yellow slant/yellow butt: glucose fermentation/ lactose and/or sucrose fermentation. Reported as A/A. -red slant/yellow butt: glucose fermentation only/ exhausted supply in slant after 12 hours, bacterium switched to peptone catabolization. Reported as K/A. -red slant/red butt or no change: no fermentation. peptone catabolized/not an Enterobacteriaceae/ no change in butt indicates organism cannot grow anaerobically. Reported as K/K or K/NC. -Cracks or lifting of agar: gas produced during fermentation of carbohydrates. Reported as +G. -Black precipitate in butt: sulfur reduction (always assume butt is yellow beneath precipitate). Reported at +H2S

After second step of nitrate reduction test:

1) formed red precipitate is a negative result. The zinc catalyzes the reduction of any remaining NO3 to NO2, not the bacteria. 2) if it remains clear this is a positive result for nitrate reduction (and denitrification and anaerobic respiration).

Two mistakes a student might make that could lead to a false+ result for the oxidase test:

1)If you wait too long to read it, it will react w/oxygen. 2) If you use a metal loop.

Describe/define a coliform:

A lactose fermenter. Gram -, oxidase -, enteric bacteria capable of fermenting lactose. Grows in the gut.

formula for aerobic respiration

C6H12O6 + 6H2O + ADP > 6CO2 + 6H2O + ATP

Organisms that have citrate permease and citrase are able to use citrate as their only source of _____.

Carbon

Catalase test

Catalase converts H2O2 (hydrogen peroxide, the substrate) into water and O2. Peroxidase converts H2O2 into H20 and a harmless non-oxygen product. Catalase is present in aerobic and facultative anaerobic bacteria and all cells of plants and animals. It's one of the most potent catalysts and is sequestered in lysosomes and peroxisomes in eukaryotes and in the periplasmic space in prokaryotes (in periplasmic space they are stored as well as destroyed by enzymes). This test detects presence of the enzyme catalase only; bubbling indicates the liberation of molecular oxygen as gas. If no bubbling, the organism is either an obligate anaerobe or breaks down hydrogen peroxide w/peroxidase. Differentiates Gram+ bacteria.

Coagulase Test

Coagulase catalyzes the conversion of fibrinogen to fibrin, forming clots; Staphylococcus does this. The formation of this causes a fibrin clot to be formed around the bacteria, protecting it from attack of WBC's. This test tests for presence of bound coagulase on surface of S. aureus cells. Uses rabbit plasma. Procedure: rabbit plasma, use inoculating loop to aseptically transfer small amount of culture to the rabbit plasma, stir 20-30 seconds. Clumping= positive result. Milky look= negative result.

Optochin Test

Differentiates alpha hemolytic Streptococci: S. pneumoniae and viridans streptococci. S. pneumoniae is sensitive to optochin. Done on blood agar.

IMViC test

Differentiates gram- Enterobacteriaceae. Determines if the enteric organism in question produces specific enzymes, can utilize alternate carbon sources for energy, or produce characteristic end products from glucose fermentation; useful for differentiating coliforms.

Bile esculin test and 6.5% salt broth- differentiating lancefield group D

Differentiates streptococci and enterococci through BEA test. BEA is selective cause it contains bile which is inhibitory to Gram+ organisms but not to enteric streptococci and enterococci. It's also differential for Group D streptococci and enterococci because it contains esculin (sugar-glucose and esculitin). Positive result is dark brown agar slant. Esculin- substrate, ferric citrate- indicator in BEA, esculitin- detectable product. Differentiates Lancefield group D.

Match the organism w/the disease it causes:

E. faecalis- UTIs and VRE infections (vancomycin resistant enterococci). S. agalictiae- neonatal sepis. S. pneumoniae- pneumonia. S. pyogenes- strep throat. S. mutans- tooth decay.

Bacitracin test

Groups A&B are both beta hemolytic and pathogenic. They can be differentiated by antimicrobial tests; pyogenes is sensitive to low concentrations of bacitracin, sensitivity demonstrated by a zone of inhibition 10mm or bigger. Resistance- no zone of inhibition or one less than 10mm. Done on blood agar Differentiates beta hemolytic streptococci

Blood Agar Test

Hemolysins: toxins produced by certain bacterial organisms that destroy RBC's and damage human tissues (they break down RBC's and release the iron). Differentiate Gram+ cocci. Blood agar plate= tryptic soy agar + 5% ovine or bovine blood. Blood agar is differential and an enriched medium used to culture these fastidious organisms. Complete destruction of RBC's= beta hemolysis, produces a lightened area in the blood agar around and underneath the colonies appearing slightly yellow and transparent. Incomplete destruction of RBC's= alpha hemolysis, partial breakdown of hemoglobin in RBC's and produces green color around/under colonies. No change= gamma hemolysis.

What hemolytic pattern is seen with Streptococcus pneumoniae? What test separates this organism from other streptococci with the same hemolytic reaction?

Hemolytic pattern- alpha. Separated by optochin test.

Mixed acid fermentation is detected by the presence of which organic molecules?

Long-term stable acids.

phenol red broth results

K: for alkaline should the pH rise due to peptone catabolism making it hot pink. - and K results indicate no fermentation. Gas bubble should be about 2mm to be considered "G". A: breaks down sugar, acid fermentation.

Motility agar differentiates bacteria on what two properties?

Motility and cellular respiration

Describe the source of electrons that enter the electron transport chain:

NADH & FADH2

The reagents added in part 1 of the nitrate test only react with and detect:

NO2

What's the catalase result for all streptococci?

Negative; Staph is +

What are two pathogenic organisms that are positive for urease production? What diseases do they cause?

P. vulgaris and E. coli. UTI's.

Explain the role of phenol red in the urea medium. How does phenol red help in the detection of a change in the medium?

Phenol red detects pH. Ammonia is basic

Urease test results

Positive- hot pink, rapid. Orange- positive reaction from a slow/weak urease producer.

Viridans streptococci

S. mutans, S. mitis, and S. salivarius

Which two streptococci have capsules?

Salivarius and pneumoniae

Mannitol Salt Agar

Selective and differential. Selects for growth of halophilic bacteria due to presence of 7.5% NaCl (used in medical microbiology to identify pathogenic and salt tolerant organisms such as Gram+ Staphylococcus species). Promotes only scant growth of Gram+, catalase + Micrococcus and Kocuria species, and inhibits growth of Gram+, catalase - bacteria in Streptococcacea except for Enterococcus faecalis. The differential ingredient is the monosaccharide mannitol which detects whether the bacteria can or can't ferment the carbohydrate. Mannitol fermentation produces acid turning the phenol red pH indicator in the medium from pink to yellow. S. aureus is part of the normal flora of the skin and respiratory tract that ferments mannitol, produces coagulase

6.5% saline Tryptic soy broth

Separates Group D into enterococci and non-enterococci. Inhibits non-enterococci (S. bovis), but selective for enterococci (E. faecalis). Only a selective medium. Positive result is turbid broth.

The catalase test is very useful for differentiating the staphylococci from the streptococci.

Staphylococci-positive Streptococci-negative

Indole Test

Substrate: tryptophan. This test detects the presence of tryptophanase by detecting indole as an end-product of the reaction involving this enzyme; tryptophanase hydrolyzes the substrate tryptophan into indole, pyruvic acid, and ammonia. Use tryptone broth which contains the amino acid tryptophan. Reagent: Indole Kovac's. Positive result has a red ring at the top.

Why is it advantageous for enteric bacteria to perform anaerobic respiration using nitrate reduction?

There's no oxygen in the digestive system.

From a biochemical standpoint, why can anaerobic organisms not survive in the presence of oxygen? What enzymes are they lacking?

They can't handle the free radicals; catalase enzyme or superoxide dismutase.

What is indicated when the bacteria growing along the stab don't turn red?

They're not going through cellular respiration

Why is it important to stab the agar when inoculating blood agar plate?

To get it away from oxygen

Superoxide radical

Toxic byproduct produced by reactions in the ETC

Methyl Red- Voges- Proskauer Tests

Two broad classes of fermentation patterns: mixed acid fermentation and 2,3-butanediol fermentation. Both Methyl Red and Voges- Proskauer tests performed in MR-VP broth which contains glucose, peptones, and buffers. These tests distinguish between coliforms by detecting fermentative end products.

When observing the results of the motility test, why do strict aerobic bacteria sometimes show inconclusive results:

We don't know if it just can't move or if it doesn't move cause there's no oxygen

Following the previous question, could you have results of the motility agar test that are positive for motility but negative for utilizing the ETC?

Yes, it would grow away from the stab line but would be clear.

Are oxidase negative bacteria capable of still performing aerobic respiration?

Yes, they just use a different enzyme on the Complex IV.

2,3-butanediol fermentation is detected by the presence of which organic molecule?

acetoin

If an organism uses the mixed acid fermentation pathway to break down glucose, the final pH in the medium will be ______, which can be detected by the pH indicator _______.

acidic; methyl red.

VP test

bacteria that engage in 2,3-butanediol fermentation. Detects an intermediate in the fermentation pathway, acetoin, that is oxidized to a red diacetyl compound in the presence of oxygen when the two REAGENTS alpha-naphthol and 40% potassium hydroxide are added. Positive result is a rose color in the upper third of the broth.

A countable plate

between 30 and 300 CFU's

Ingredients that make MacConkey agar a selective medium? What do they select for?

bile and crystal violet. Gram- enterics

Chemical (gaseous) agents

bind nucleic acids and proteins together that inhibits cell metabolism. Ethylene oxide

Surfactants

break the surface tension on liquids (that holds liquids together). Lysol

Tests determining Gram+ bacteria:

catalase, mannitol salt agar, coagulase, blood agar, bile esculin agar, 6.5% NaCl broth, bacitracin and optochin tests

Treponema pallidum

causative agent of syphilis

Phenol and the derivatives of phenol called phenolics:

cause breakdown of cellular proteins and/or disrupting the cytoplasmic membrane. Clove oil

Antimicrobials (chemotherapeutic agents)

chemicals used to decrease pathogens internally

What two groups of bacteria can be differentiated by the utilization of the ingredients above?

coliforms and lactose non-fermenters

MR test

coliforms that utilize the mixed acid fermentation pathway. Acids produce drop the pH. pH indicator: methyl red. After added, positive result will be red. Detects the presence of long-term stable acid products.

Oxidase negative bacteria lack a specific protein known as:

cytochrome c oxidase

Halogens

denatures proteins and include the oldest antimicrobials iodine and chlorine.

Sterilants

destroy ALL microbes, their toxins, and spores. Too toxic to be used in direct contact w/humans or animals but can be effective when sterile equipment or facilities are required.

Oxidizing agents

destroy proteins, specifically cellular enzymes. Oxidize cellular enzymes, making them nonfunctional, ultimately interfering w/metabolism and killing the cell. Hydrogen peroxide

Oxidase test:

detects for cytochrome c oxidase on Complex IV of the ETC. Indicator- redox, chemical dye that changes colors when it becomes oxidized. Reagent- phenylenediamine, colorless in its reduced form. If cytochrome c oxidase present, phenylenediamine becomes oxidized and cytochrome c oxidase becomes reduced. In oxidized state, phenylenediamine turns dark purple. Family Enterobacteriaceae- large group of Gram- bacteria that are all oxidase-. Oxidase + include Alcaligenes and Neisseria. Observe reaction within 30 seconds because it will eventually react w/oxygen in the air causing it to oxidize (false +). Procedure: add 2-3 drops of reagent to filter paper, use wooden stick to transfer bacteria on top of it, observe for 30 seconds.

Motility test

differential test that has a broad application across many groups of bacteria. Allows us to differentiate between Enterobacter cloacae and Klebsiella pneumoniae. This test gives us information about 1) ability to move through a watery medium and 2) the respiratory status of the isolate- whether the tested bacteria are producing ATP by cellular respiration. As the nutrient level drops, motile bacteria move away from the stab line in search of nutrients. TTC is a colorless molecule that's a competitive inhibitor of the electron transport chain. Therefore, if the organism growing in the motility agar is using cellular respiration to make ATP, TTC steals the electrons (is reduced) and will form formazan, the red-colored molecule. TTC allows us to determine the type of metabolism used by bacteria (cellular respiration- turns red) and if the organism is motile.

nitrate reduction test

differentiates bacteria and provides information about multiple metabolic processes at the same time. Uses nitrate reagents A: sulfanilic acid and B: alpha-naphthylamine, as well as zinc dust. After first step, if the bacteria in the culture have reduced NO3 to NO2, the nitrate reagent A converts NO2 into a diazonium salt, reacting with the alpha-naphthylamine to form a red precipitate. *the reagents only react with and detect nitrate (NO3). The red precipitate indicated the organism has nitrate reductase and is positive for nitrate reduction and anaerobic respiration.

Alcohol

dissolves lipids in the cytoplasmic membrane and denatures proteins. Isopropyl

Three steps of cellular respiration:

glycolysis, the Krebs cycle, and the ETC

Aldehydes

inactivate proteins and nucleic acids

Heavy metals

inactivate proteins by binding w/the sulfhydryl groups on the active site of proteins and include mercury, copper, and silver.

The presence of the enzyme tryptophanase is indirectly detected by the waste product _____ reacting with ________ reagent.

indole; indole-Kovac

What's the ingredient in MacConkey agar that makes this media differential?

lactose and neutral red

WBC's

location in bacterium where toxins stored/destroyed by enzymes

Urease test

many Gram- bacilli are able to utilize this waste product as a source of nitrogen; they produce urease, which hydrolyzes the carbon-nitrogen bond in urea resulting in production of carbon dioxide and ammonia. Phenol red: pH indicator. If organism produces urease and can hydrolyze urea as a nutrient source, ammonia accumulates raising the pH. Once the pH reaches 8.4, the phenol red changes from pale orange to hot pink. This test can also distinguish rapid urease positive organisms from slow/weak urease positive organisms. Many enteric bacteria are urease positive, but the only rapid urease positive genus is Proteus. Substrae: urea Enzyme detected: urease Detectable product produced: ammonia

Tests determining Gram- bacteria:

oxidase, macconkey agar, triple sugar iron agar, urease, IMViC

NAD+

oxidized form of nicotinamide adenine dinucleotide

phenol red

ph indicator in phenol red broth. Broth starts pink/peach color. If acid is produced (organism ferments the particular carbohydrate in the tube), broth turns yellow. Nutrient broth contains peptone and has a neutral pH (7.2-7.4). Peptones serve as an energy source, carbohydrates serve as nutrition and energy source.

Tests that identify both Gram+ and Gram- bacteria:

phenol red broth, nitrate, and motility agar

Clear broth after addition of reagents A&B, and zinc powder:

positive for anaerobic respiration and denitrification, negative for nitrate reduction.

Red precipitate observed after reagents A&B only added:

positive for anaerobic respiration and nitrate reduction, negative for denitrification.

NADH

reduced form of nicotinamide adenine dinucleotide

subject of the nitrate reduction test

soil-dwelling, facultative anaerobic bacteria. They complete the nitrogen cycle in a process called denitrification in which NO3 is converted to N2.

If after first step of nitrate reduction test the precipitate remains clear:

the bacteria either 1) couldn't utilize nitrate as an alternate electron acceptor or 2) they completed denitrification, producing N2 gas which left the tubes via the holes in the cap.

cytochrome c oxidase (nitrate reduction test)

the last component of the electron transport chain that facilitates the transfer of low-energy electrons to oxygen (in aerobic cellular respiration)

If reagents A&B added to an uninoculated tube:

the tube will turn red

Antiseptics

used on living tissues. Scope, povidone (iodine), hand sanitizer, hibiclens

Disinfectants

used on non-living surfaces. Clorox, lysol, cidex (ammonia), and alcohol.

Macconkey agar test:

used to differentiate members of the family Enterobacteriaceae, gram- mostly enteric bacteria. Selective ingredients: 1) bile salts which inhibit any non-enteric gram- bacteria. 2) crystal violet which along w/bile salts inhibits growth of most gram+ bacteria. pH indicator: neutral red. If pH is 6.8 or higher, neutral red will remain colorless, but if pH drops below 6.8 (acidic), neutral red turns red. These Enterobacteriaceae are divided into coliforms and lactose nonfermenters. Coliforms: indicators of fecal contamination and produce acid from lactose fermentation (colonies turn red). Two genera that typically precipitate bile salts are Escherichia and Citrobacter. Coliforms that produce a capsule and utilize the lactose in the medium have heavily mucoid colonies. Because these capsules are composed of carbohydrates, they don't ferment all of the lactose resulting in only the outer edge of the colony being red (Klebsiella and Enterobacter). Reversion can occur if left in incubator past 24 hours.

Citrate test

uses Simmon's citrate agar to test for the ability of organisms to utilize citrate as the sole source of carbon. If citrate is utilized, the organism must have the enzyme citrate permease which facilitates the transport of citrate into the cell. Once citrate is inside the cell, they must have another enzyme, citrase, to oxidize this substrate. During this oxidation, sodium carbonate (alkaline) forms causing a rise in pH. To detect the change, bromthymol blue is included in the medium. Positive result is blue.

Dilution factor

volume of original/total volume