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SSP Micro Lab 6

Terms in this set (54)

How do microorganisms modify their environment?
utilize chemicals in solution as building blocks for growth, energy sources, and reproduction
Extracellular
enzymes that are found outside the supernatant culture media
Intracellular
enzymes when only death or disruption of the cell wall causes their release from the cell
Constituitive
enzyme that is constantly produced in a cell
Adaptive
enzymes that are produced only in response to the presence of their specific substrate
How are intestinal pathogens identified
by their ability to ferment various sugars
Coliform organisms
identified by the presence or absence of acetyl-methylcarbinol in the fermentation of glucose
Proteolytic Clostridia
identified from gelatin liquefaction
Proteus organisms
differentiated from the gram-negative intestinal which are urease negative
Other diagnostic techniques
production of indole, hydrogen sulfide, hydrogen, ammonia, organic acids
Carbohydrate Utilization (acid and gas formation)
main gasses that breakdown
carbon dioxide and hydrogen
Carbohydrate Utilization (acid and gas formation)
name of inverted vial
Durham tube, gas is collected if it is formed during growth
Carbohydrate Utilization (acid and gas formation)
components of the media
sugar, phenol red (pH indicator)
Carbohydrate Utilization (acid and gas formation)
Phenol Red
red at pH 6.8, yellow at pH 5.2 (Acid pH)
Carbohydrate Utilization (acid and gas formation)
used which bacteria
E. coli, E. faecalis, B. Cereus, Staph Simulans
Carbohydrate Utilization (acid and gas formation)
observations
growth is usually seen by observing turbidity in the tube, yellow is acid production and bubble show gas production
Starch Hydrolysis
what is starch
a complex carbohydrate or polysaccharide
Starch Hydrolysis
quantitive test for starch
appearance of blue color w addition of a solution of iodine
Starch Hydrolysis
cleavage products when starch is hydrolyzed
dextrins, maltose, and glucose
no longer give color to the reaction
Starch Hydrolysis
procedure
using starch agar dish streak down the middle
E. coli (-) and b. Cereus (+)
streak down the middle to create a central zone, area of contrast when we develop the plate w iodine
Starch Hydrolysis
testing for starch hydrolysis
invert plate over iodine crystals, iodine vapor will react with the unhydrolyzed starch present in the plate and will produce a blue/purple color in areas where starch has not been broken down by the organism. clear zone around area of organism will indicate hydrolysis of the starch in the agar
Casein Hydrolysis
shows how microorganisms can utilize amino acids
what is casein
principle protein in milk, exists as a colloidal suspension giving milk an opaque look
what is protein breakdown called
peptonization
Casein Hydrolysis
what bacteria is used
B. cereus (+) and E. coli (-)
+ = clear zone
- = no clear zone
Gelatin Hydrolysis
what is gelatin
a protein secured byt he hydrolysis of collagen, a component of connective tissue and tendons of animals
Gelatin Hydrolysis
medium
- will remain liquid after is has been utilized
- do not agitate while in liquid condition because liquefication occurs slowly from the surface down and agitation will mix hydrolyzed with the unhydrolyzed gelatin = false negative
Gelatin Hydrolysis
what bacteria are used
E. Coli (-) , B. Cereus (+) , E. Faecalis (+) , P. Vulgaris (-)
Gelatin Hydrolysis
testing for hydrolysis
place tubes in ice water after being incubated, negative tubes will solidify, hydrolyzed will remain fluid
Blood Agar
what is hemolysis
the breakdown of the membrane of red blood cells
Alpha hemolysis
partial hemolysis, appears green
Beta hemolysis
complete hemolysis, appears clear
Gamma hemolysis
no hemolysis, no color changes
Blood Agar
components of the agar
Trypticase Soy Agar (TSA) and 5% sheep's blood
Acetylmethylcarbinol Production and the Methyl Red test
two test that can be performed on the same medium, characterization of gram-negative nonspore forming rods
what is a test testing for AMC commonly called
Voges-proskauer reaction, VP test
AMC (VP) and MR used what bacteria
E. coli and E. cloacae
AMC (VP) and MR tubes
contain 5 mL, establishes a ratio of a larger volume of air in the test tube to a small volume of medium, condition which promote AMC production
AMC (VP) and MR Day 4
Agitate test tubes
remove 1 mL of tube A and place it into a clean test tube labeled test tube B, save 4mL left over for MR procedure,
add to 1mL tube- 15 drops of alpha-naphthol solution then add 5 drops of 40% KOH solution, after 5 min results present
AMC (VP) and MR results
VP positive = medium turns red
VP negative = yellow or copper color of medium
MR results
test for acid production by adding 1-2 drops of an alcoholic solution of methyl red, immediate results

MR positive = red color (pH of 4.4)
MR negative = yellow color (pH of 6.0)
Indole production
what is indole
indole is a nitrogen containing compound formed from the degradation of the amino acid tryptophan by various bacteria
Indole production
importance
only certain bacteria form it and that it can be readily detected chemically, suitable as another differentiating reaction
Indole production
component in the medium
tryptone-a digestion product of certain proteins, used as a substrate because it contains tryptophan
Indole production
tubes
1% tryptone w E. coi and E. cloacae
15 tryptone broth/ 1% glucose w E. coli
Indole production
indole test
presence of indole using Kovacs method, add 250 ul of Kovacs solution to the 6ml culture fluid (contained in a vial and must be cracked between thumb and pointer finger before dispensing drop by drop)
rotate tube between hand or on lab table
Indole production
results
positive result = reddening of the alcohol layer
in tube w 1% glucose, rapid acid production from the utilized carbohydrates inhibits the induction of enzymes which attack protein
"Protein sparing action of carbohydrate"
Citrate test
completed using Simmons citrate agar to differentiate the gram negative bacteria on the basis of citrate utilization

organisms which can utilize the citrate will produce an alkaline reaction
Citrate test
indicator
bromthymol blue, in the presence of alkaline conditions the indicator will change from green to blue
Citrate test
results
positive- indicted by the bromthymol blue indicator changing green to blue
Hydrogen sulfide production
media
must contain salts of metal such as bismuth and iron
contains lactose and dextrose 10:1
thiosulfateiron (klinger iron agar)
Hydrogen sulfide production
darkening along line of the stab
caused by formation of h2s
Hydrogen sulfide production
results
-yellow slant from fermentation of the dextrose, once exhausted the acids are oxidized and slants goes back red
- yellow coloring to red coloring does not happening without oxygen and therefore does not happen at the butt of the test tube and stays yellow
- organisms that cannot ferment dextrose or lactose will remain completely red
Catalase activity
what is catalase
an enzyme found in most bacteria
catalyzes the breakdown of hydrogen peroxide with the release of free oxygen gas
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