Lab Test 3 Flashcards
What are exoenzymes
Enzymes secreted out of the cell to act upon a large substrates (macromolecules) in the environment
ex. hemolysin
Endoenzymes
Enzymes whose substrate is located within the cell to perform a reaction. such as = ATP production, creation or repair of cell parts, creating movement, DNA transcription, replication, and translation.
How do bacteria feed
By diffusion, secreting exoenzymes into the watery medium surrounding them, and then absorbing the dissolved end products through the cell wall and membrane.
-dependence on water is a weakness - usually why dried foods can resist bacterial spoilage without the need for preservatives or refrigeration.
Exoenzyme reactions are classified as _______ reactions because they use water to split the large molecules
Hydrolytic
The substrates of exoenzymes are typically _____
Large, polymeric macromolecules such as carbohydrates, lipids, and proteins of various sorts.
- These molecules are usually poorly soluble in water, existing as suspensions which give the watery medium a turbid, opaque appearance.
- End products of exoenzymes are small enough to diffuse through the membrane and are usually soluble making solutions containing them clear.
The substrates of exoenzymes give a ___ appearance to a medium
Cloudy
The end products of exoenzyme activity give a _____ appearance to a medium
Clear
Which organism would produce more exoenzymes: one that lives in the intestine or one that lives in dirt
Dirt - organisms living in intestine don’t need to use many exoenzymes because usually food is already broken down for them via hosts enzymes
What is the enzyme that digests the substrate starch?
Amylase splits the starch into disaccharides (maltose), then maltase splits maltose into individual glucose molecules
What is the indication of a positive test for starch hydrolysis & what is the reagent used
Learning of medium; Iodine
- Bacteria is grown on a plate with starch incorporated into the agar during preparation. The substrate is opaque, but is the exoenzymes amylase and maltase are produced and released into the agar, an area around the streak of growth will show a clearing
- Iodine turns purple in presence of starch but nothing happens with just maltose or glucose
What bacteria is the starch hydrolysis test useful for?
All different types of bacteria
What substrate does amylase work on? Maltase?
Amylase = starch Maltase = Maltose
What substrate does Lipase work on?
Triglycerides
What is/are the end products of lipid hydrolysis
Three fatty acids and a glycerol
Indication of a positive lipid hydrolysis test, reagents? useful for?
clearing of medium (spirit blue agar plate used), no reagent used
- triglycerides = insoluble, but glycerol and FA = soluble
- a lot of bacteria
What is the substrate that caseinase works on
Casein
What are the end products of casein hydrolysis
Amino acids
Indication of a positive test: casein hydrolysis, reagent used, useful for
Clearing of medium (milk plate)
- The insoluble polymer, casein, is cloudy in suspension, but the amino acids produced by its breakdown by the enzyme casinos are soluble, and therefore clear in solution
- no reagent used
- useful for a lot of bacteria
What substrate does the enzyme gelatinase work on?
Gelatin
What is/are the end product (s) of gelatin hydrolysis
Amino acids
Indication of a positive test - gelatin hydrolysis, reagent, useful for
Liquid appearance: Inoculating bacteria into a deep tube using gelatin as the solidifying agent. After incubation if the bacteria have digested the gelatin, the medium will be liquefied
- no reagent
- useful for a lot of bacteria
Fermentation
Is an anaerobic energy producing process utilized by anaerobes, facultative organisms, and some microaerophiles to produce ATP.
Why is testing for fermentation of a single sugar not useful?
For most aerobes, fermentation isn’t an important process therefore performing fermentation tests would be useless. Strict anaerobes ferment all sugars so they are not easily differentiated by which sugars they ferment giving positive results for all.
Any media used to detect carbohydrate fermentations must have the following features:
Only one carbohydrate, a pH indicator, a durham tube (to catch any CO2 gas produced during the fermentation process)
Carbohydrate fermentation reaction
Carbohydrates (glucose, sucrose, lactose) —fermentation enzymes —> organic acids, alc, CO2, H2
Indication of a positive test for carbohydrate fermentation, reagent, and organisms it is most useful for
Color change from purple to yellow, from acids produced (pH indicator), and bubbles in durham tube**
- most useful for facultative organisms
- *no bubbles is a NEGATIVE test, even if there is a color change
Two important pathogenic genera of gram positive cocci
Staphylococcus and streptococcus
What is the substrate for the enzyme catalase?
H2O2
Catalase Test
- done to separate the genera of gram positive cocci
- strep (catalase -)
- staph and micrococcus (catalase +)
- a positive test is not definitive for staph
What are the end products of catalase
H20 and 02
Indication of a positive catalase test and what organisms is it useful for
bubbles
- confirm or rule out strep (-)
- suggest staph or micrococcus (+)
Mannitol reaction
Mannitol —digestive enzymes–> acids
-color change from red to yellow, suggests staph a.
What is the most definitive test for determining staph
Coagulase, only pathogenic staph produce this enzyme
Coagulase test reaction, indication of a positive test and what is the test useful for
Fibrinogen (liquid) —–coagulase–> fibrin (solid)
- solidification
- definitive for staph a.
Species of streptococcus are separated from one another by…
hemolytic activity and lance field serological groupings
Strep pyogenes
Causative agent of “strep throat”
Hemolysis
The splitting of red blood cells, an enzymatic reaction carried out by some organisms to feed from the highly nutritive contents of the cells, or to reduce oxygen concentration in the surroundings
Beta hemolysis
Complete clearing of blood agar
-Cause not only the rupture of the red blood cell membrane, but also the total digestion of the hemoglobin released from the cell into its iron and amino acid building blocks
Alpha hemolysis
Incomplete clearning or greening of blood agar
- break open red blood cells but do not completely digest the hemoglobin contained inside
- instead hemoglobin is modified to form hemoverdin, a greenish compound
Gamma hemolysis
No clearning
- non-hemolytic and do not lyse RBC’s.
- these organisms tend to not be pathogens
Beta hemolysin reaction, indication of a positive test, what organism does it indicate
Hemoglobin —beta hemolysins–> amino acids + Fe
- complete clearing
- strep pyogenes
Alpha hemolysin reaction, indication of a positive test, what organism
Hemoglobin —-alpha hemolysins—> hemoverdin
- incomplete clearing/greening
- strep pneumoniae
What are the end products of beta hemolysis
Amino acids and iron
End products of alpha hemolysis
Hemoverdin
Lancefield serological groupings
Group streps. based on antigens on their cell surface separate from the ability to cause hemolysis
Oxidase test
- oxidase is one of the enzymatic components of the electron transport chain
- all aerobes use the ETC and have oxidase; all anaerobes lack the enzyme, since they use fermentation
What organisms is an oxidase test definitive for?
Pseudomonas and neisseria
Oxidase test, indication of a positive test, reagent, organisms
Hydrogen (NADH) & Oxygen (air) —cytochrome oxidase—–> water
- color change from pink to purple to black within 60 seconds (+)
- no color change, or a color change to black after 60 seconds is a negative reaction
- helpful at determining neisseria
What do the SIM stand for in SIM medium
Sulfur, iodine, motility
-has ingredients to indicate: motility, indole production and H2S production
SIM Medium
- contains agar (semi-soft)
- hard enough that bacteria will be held in place if they don’t have flagella
- soft enough that bacteria with flagella will be able to move throughout the agar
What are the sulfur containing substrates in a sIM medium
cysteine, and thiosulfate
-indicator for the reaction = iron
Reaction where thiosulfate is the substrate, indication of a positive test, and what organisms useful for
Thiosulfate —thiosulfate reductase—> sulfite + hydrogen sulfide gas
- Black solid (FeS)
- Gr negative enteric rods
Reaction where cysteine is the substrate, indication of a positive test, and what organisms useful for
Cysteine —cysteine desulfurase—> pyruvic acid + hydrogen sulfide gas + ammonia
- Black solid (FeS)
- Gr negative enteric rods
Four possibilities for results in a SIM tube
- Cloudiness throughout: motile, - for H2S production
- Black throughout: motile, + for H2S production
- Cloudiness along stab line: non motile and - for H2S production
- Black along the stab line: non-motile & positive for H2S production
What are the three sugars in the triple sugar iron agar reactions
Glucose, lactose, and sucrose
What is the TSIA used for
To differentiate between facultative organisms, by showing the pattern of which of the three common sugars are fermented to produce ATP when in low oxygen environments. The TSIA medium, since it is a solid agar slant which is stabbed as well as streaked is anaerobic in the lower portion of the stab and aerobic near the surface of the slant
What can the TSIA medium determine
- Acid production from glucose fermentation only
- Acid production from lactose and/or sucrose fermentation
- gas production from fermentation of sugar
- and the release of H2S from thiosulfate or cysteine
Why is glucose in a smaller amount in the TSIA medium
All bacteria capable of fermenting sugars will selectively act upon glucose first, since it is the simplest most widespread sugar. Only after exhausting the supply of glucose will bacteria switch to the fermentation of other sugars. Limiting the glucose concentration forces the bacteria to switch.
What will the TSIA tube look like if only glucose was fermented
The tube will change yellow initially, but then the neutral/basic end products of respiration of sugars and proteins occurring at the top will turn the slant portion red.
- neutral from sugars
- alkaline from proteins as AA’s release ammonia
What is the pH indicator in the TSIA tube
Phenol red
What will a TSIA tube look like if glucose and one of the other two sugars was fermented?
First the tube will be all yellow but after 48 hours the slant will be red.
*Fermenters of multiple sugars will yield a false negative result for sucrose/lactose fermentation if recorded after 24 hours.
What does a control TSIA tube look like?
All orange throughout
TSIA tube where no fermentation has occurred
Red slant and orange throughout, after 48 hours turns all red
What is an end product that always occurs with fermentation
Gas! If no fermentation occurs there can be no gas produced beneath the surface
If a TSIA tube appears blackened what is going on
If the bacteria inoculated produces thiosulfate reductase or cysteine desulfurase, H2S gas is released in the agar, and will combine with iron to form FeS (black precipitate) as it rises to the top
A yellow butt is ____ whereas an orange butt is ____
acidic; basic
What are the tests that make up the iMViC series
Indole test, methyl red, voges proskauer test and the citrate test
What is the substrate for tryptophanase
AA tryptophan
Indole test & reaction
-E. Coli produces tryptophanase thus yielding a positive reaction
-indole = only unique end produce of the action of the enzyme
Tryptophan —–trypotophanase—> indole + pyruvate + ammonia
Indication of a positive indole test, recent and organisms
Cherry Red layer at the top, Kovac’s reagent, and to differentiate E. Coli from enterobacter and other lactose fermentors apart
Methyl red test and reaction/indication of a positive test/reagent/organisms
-Indicates extreme acids produced (like ph 3-4) from fermentation of pyruvate to mixed acids such as: lactic, acetic and formic
glucose—fermentation enzymes–> strong acids
Red, methyl red
-To differentiate E. Coli from enterobacter and other lactose fermenters
Voges proskauer test
Some glucose fermenters produce less acidic end products (enterobacter) during their fermentation process (ph 6). Therefore the VP test is designed to test specifically for the end product acetylmethylcarbinol
-VPA and VPB are added to the glucose broth after incubation and will react with acetylmethylcarbinol
VP test reaction/indication of positive test/reagent/organisms
Glucose –fermentation enzymes–> 2,3 butanediol
brick red; VP reagents A &B
-differentiate E. coli from enterobacter and other lactose fermenters apart
Citrate test
-Enterobacter has citrate but not E.Coli therefor enterobacter can survive in a medium containing only citrate whereas E. Coli cannot
Simmons citrate medium
- contains inorganic material rather than organic nutrients
- only carbon chain is therefore citrate and if an organism cannot use citrate it cannot grow
Citrate reaction/positive test/reagents/organisms
citrate —citrase–> Pyruvate + Co2
- blue color change due to excess Co2
- to differentiate E.Coli from enterobacter and other lactose fermenters apart
The accepted poor water quality pathogen is __ & why?
E.Coli; since E. Coli is found in all human and some animal waste, but never in nature, if E. Coli is in the water, feces contamination has occurred and the water is determined to be undrinkable
What does the water analysis do
Tests for coliforms = gram - rods such as E. Coli that are common in intestinal tract
Quantitative water analysis
Involves the filtration of water which collects all bacteria from the water. The filter is disassembled and the filter is placed on a petri dish of EMB liquid a
Drawbacks of quantitative water analysis test
- costly and time consuming
- apparatuses are expensive
- aseptic techniques require trained personnel
What are the three parts of the quantitative water test
- presumptive test; involves innoculating measured amounts of water into 3 lactose fermentation broths. Presence of acid (turning tube from purple to yellow) and gas in any one of the tubes is a positive presumptive test and warrants progression to next test
- Confirmed test; used to confirm that E coli is present in the water sample. One of the lactose broths is used to streak an EMB plate for isolation. Greenish gold colonies = positive test, no greenish gold just means some other organism
- Completed test; double check. A greenish gold isolated culture is grown and a gram stain must be done to indicate small, gr- bacilli. Lastly a new tube is innoculated and must show acid and gas production
Serology
The study of serum, the clear, non-cellular fluid component of the blood
Seriological reactions
Use antigen-antibody specificity to identify antigen/pathogen involved
-since antibodies selectively bind only to the antigen which solicited ints production the specificity of the binding can be used in clinical settings to either identify the antibody type found in a patients serum or identifying antigens
Antigens from bacteria
- proteins of the cell wall
- cell membrane
- flagella
- pilli
- fimbrae
- exoenzymes
- exotoxins
- capsule
Antigens from viruses
-proteins of the capsid or peplomer proteins
antigens from protozoa
-worms/yeasts: cell surface proteins
Antigens from our own bodies
proteins made during usual or disease conditions not normally present in the body such as HCG found in the urine during pregnancy
3 main categories of serological tests
precipitation
agglutination
enzyme linked
Precipitation
The combination of soluble antibody and soluble antigen best seen in a gel immuno-diffusion plate also called as Ouchertolony plate
- A thick agar plate has wells cut into it and into a central well is placed the unknown component and known potential matching components placed in wells arranged in a circle around the center
- the soluble components diffuse through the agar and when they meet the binding is visible as a faint milky haze
Agglutination
Occurs when the antigen is part of the surface of a cell and when antibodies bind the entire cell is bound to the antibody
-ex. blood typing in humans
clumping with the addition of antibodies against antigen B means they have B type blood, clumping with both antibodies means AB, clumping with none means blood type O
Titer
A rough measurement of how much antibody is present in a patient’s body
-a high titer indicates a more extreme response and thus a more serious infection than a lower titer
Enzyme linked immunoabsorbet serological assay (ELISA)
Involves a color change to indicate antigen-antibody binding
- in ELISA tests the known component prepared in the lab is joined to an enzyme capable of a color change
- common tests = pregnancy and HIV tests
Why are homogenization/dilution necessary
Homogenization necessary to eliminate possibility of uneven distribution of bacteria on surfaces of food
Dilution necessary to get a countable plate (30-300) when sampling foods if no dilution then colonies will be too close together
Cheeses that are mold ripened from the inside out by penicillium roqueforti
Blue cheese, roquefort
Cheeses red smear ripened from outside in by a mixture of brevibacterium and yeasts
Munster, Limberger
“eyes” from Co2 bubbles in hardened curd
Swiss, havarti, gouda, gruyere
Acid curd cheeses (strep lactose and lacto bacillus)
Cottage cheese, cream cheese
Longest curing time for a hard cheddar cheese
Extra sharp cheddar
Mold ripened from outside in by penicillium camemberti
Camembert (brie if made anywhere other than france)
