Exercises 14-22

Blood Agar

What media type tests for the presence of exoenzymes?

Gelatin

Polymer of collagen that makes up connective tissues

• Liquefies above 25ºC

Gelatinases

Break down collagen

• Nutrient acquisition, virulence

Embden-Meyerhof Pathway (Glycolysis)

Converts 1mol glucose to 2mol pyruvate

Aerobic Respiration

Molecular oxygen as the final electron acceptor (more ATP generated)

Anaerobic Respiration

Inorganic ions other than oxygen are final electron acceptor

Fermentation

Doesn’t require oxygen and organic substrate is final electron acceptor

Phenol Red

Indicator of acid production (yellow color = acidic)

Durham Tube

Indicator of presence of air bubble

Indole Test

Measures ability of bacteria to break down tryptophan to indole via tryptophanase

Methyl Red Test

Determines ability to oxidize glucose to make acid end products

Voges Proskauer Test

After enough time, K. aerogenes converts acids to acetoin

Barritt’s Reagent

Turns pink with acetylmethylcarbinol

Citrate Test

Determines the ability of an organism to use the enzyme citrase to use citrate as a sole carbon source

Bromothymol Blue

Used in the Citrate Test

• Indicator → turns from green to blue at a pH above 7.6+

Citrate Carbon Source

If there is growth present on the slant, and if the color has changed from green to blue

Hydrogen Sulfide

Has the same medium as Indole test

• Gas produced by certain Enterobacteriaceae by two pathways

Cysteine Desulfurase

Reduction of amino acids that contain sulfur lie cysteine (found in peptone) using this in pathway 1 of H₂S gas production

Thiosulfate Reductase

Reduction of inorganic sulfur compounds using this in pathway 2 of H₂S gas production

Urease Test

Designed principally to distinguish Proteus, Providencia, and Morganella from other members of Enterobacteriaceae

• This test can yield ammonia and carbon dioxide

• Ammonia production indicated by magenta color

Complete Proteolysis

Indicating elevated pH due to deamination

Acid Clot

Indicated by solidified medium with pink acid top and reduced acid bottom

Curd Formation, Alkaline Reaction

Yellowish semisolid bottom with a blue top

• Partial degradation of casein into shorter polypeptide chains

Alkaline Conditions

Purple litmus reaction with reduced litmus at the bottom

Acid Production

Pink tube without acid clot formation or proteolysis

Anaerobic Respiration

Where nitrate can serve as a terminal electron acceptor, and ther eduction of nitrate is catalyzed by nitrate reductase

Catalase Test

Performed to distinguish members of the Staphylococcaceae and Micrococcaceae species from Streptococcaceae

Bacillus

Gram-positive rods that are positive for the catalase test

Clostridium

Negative for the catalase test

Oxidase Test

Used to differentiate strict aerobic respiratory microorganisms from facultative or fermentative microorganisms

• More commonly, used to differentiate Enterobacteriaceae (negative) from Pseudomonadaceae (positive)

• Can be used for identification of Neisseria (positive)

Cytochrome Oxidase

Heme-containing enzyme that is the terminal redox protein used in the ETC of microorganisms that use oxygen as their terminal electron acceptor

• Responsible for transferring electrons directly to oxygen to form water as one of the products of aerobic respiration

Chromogenic Reducing Agent

This is used in the cytochrome oxidase test as an indicator to detect bacteria that produce cytochrome oxidase

• Chemical that changes or produces color as it become oxidized

p-aminodimethylaniline oxalate

Donates electrons to cytochrome oxidase and becomes oxidized itself, changing colors from light pink to a dark maroon (almost black)

• Indicator used in lab for the Oxidase Test

Mixed Acid Fermentation

The methyl red test indicates whether or not the bacteria utilizes what

Hydrolytic Enzymes

Exoenzymes are mainly this → reduce high-molecular-weight materials into their building blocks by introducing water into the molecule, liberating smaller molecules, which may then be transported into the cell

Endoenzymes

Function inside the cell and are mainly responsible for synthesis of new protopalsmic requirements and production of cellular energy from assimilated materials

Amylase

Enzyme involved for the hydrolysis of starch → results in maltose molecules

Lipases

Hydrolyze triglycerides

• Cleave the ester bonds in triglycerides by the addition of water to form the building blocks glycerol and fatty acids

Kovak’s Reagent

p-dimethylaminobenzaldehyde, HCl, and butanol combined form this

Litmus Milk

Differential medium in which microorganisms can metabolize milk substrates depending on their enzymatic complement

Casein

Milk protein

Acid Curd

Lactic acid (or other organic acid) cause the precipitation of casein as calcium casinate to form an insoluble clot

• Hard clot, will not move

Rennet Curd

Paracasein converts to calcium paracaseinate (in the presence of calcium) and forms an insoluble clot

• Soft semisolid clot

Rennin

Enzyme that acts on casein to form paracasein

Zinc

Will catalyze the spontaneous reduction of nitrate to form nitrite

• Will only cause the medium to turn red if in the presence of UNCONVERTED NITRATE

SIM Media

Differential medium used to detect three bacterial characteristics:

• Sulfide production

• Indole production

• Motility

Commonly used to differentiate members of the Enterobacteriaceae family

IMViC Tests

Special tests for the separation of enteric microorganisms

• Indole, Methyl Red, Voges-Proskauer, and Citrate

Gelatin Agar

Agar that is primarily used as a growth medium to identify bacteria that produces gelatinase enzymes

• Also used for general microbial growth and for identifying Vibrio species

Tributyrin Agar

Agar that is primarily used to detect and identify bacteria capable of producing lipase

• Needs a substrate to determine results

Starch Agar

Agar that is primarily used to detect and identify microorganisms that can hydrolyze starch—in specific, those that can produce α-amylase

Gelatinase Test

Test that involves inoculating a nutrient gelatin medium with a bacterial culture, incubating it, and then checking for liquefaction

Starch Hydrolysis

Test that involves flooding an inoculated starch agar plate with Gram’s iodine solution

Exoenzymes

Enzymes that act on substances outside of the cell—most high-molecular-weight substances are not able to pass through cell membranes and use be degrades into low-molecular weight materials before being transported into cell