Lab Practical 2 Review

Question 1

definition of SELECTIVE MEDIA

Answer 1

where ONE OR MORE INGREDIENTS that begin to INHIBIT the GROWTH of UNWANTED BACTERIA while encouraging the GROWTH of DESIRED ONES

Question 2

definition of DIFFERENTIAL MEDIA

Answer 2

helps to DISTINGUISH one MICROORGANISM TYPE from another that is GROWING ON THE SAME MEDIUM

• the type of MEDIA that uses the BIOCHEMICAL CHARACTERISTICS of a MICROORGANISM that begins to GROW IN THE PRESENCE OF SPECIFIC NUTRIENTS/INDICATORS that are ADDED TO THE MEDIUM
• allows to VISIBLY INDICATE the DEFINING CHARACTERISTICS of a MICROORGANISM

ex. the BLOOD AGAR PLATES

Question 3

can a MEDIA be both DIFFERENTIAL and SELECTIVE?

Answer 3

YES!

ex. the MACCONKEY MEDIA–contains LACTOSE, BILE SALTS, and NEUTRAL RED DYE

• BILE SALTS; helps to INHIBIT the GROWTH OF GRAM+ ORGANISMS – is SELECTIVE FOR GRAM NEG BACTERIA (selective)
• LACTOSE; with the ADDITION of NEURAL RED DYE– COLORLESS at pH 6.8 + RED at LOWER pH
• if we have LACTOSE FERMENTATION; have COLONIES TURN PINK OR RED :)

Question 4

describe the EOSIN METHYLENE BLUE AGAR

Answer 4

• contains LACTOSE and DYES EOSIN Y & METHYLENE BLUE
• SELECTIVE for GRAM-NEGATIVE BACTERIA (the specific DYES INHIBITS the GROWTH of GRAM + BACTERIA)
• DIFFERENTIATES for LACTOSE FERMENTERS from NONFERMENTORS

RESULTS:

(+) IF WE HAVE LACTOSE FERMENTATION;

• colonies turn DARK PURPLE or BLACK
• due to ACID END PRODUCTS OF LACTOSE FERMENTATION
• can also test HOW WELL AN ORGANISM FERMENTS LACTOSE – affects the COLOR OF COLONIES (if it is less effective – colors is more pinkish or purplish color)

Question 5

describe MANNITOL SALT AGAR

Answer 5

• has a very HIGH SALT CONCENTRATION (around 7.5%) and is SELECTIVE FOR GRAM-POSITIVE COCCI
• has MANNITOL (as CARBON SOURCE) + PHENOL RED INDICATOR
• DIFFERENTIATES between PATHOGENIC and NONPATHOGENIC STRAINS

conditions of PHENOL RED INDICATOR:

• PINK/RED: pH > 7
• YELLOW: pH < 6.8

RESULTS:
- looking for MANNITOL FERMENTATION!!
- if POSITIVE FOR FERMENTATION; turns YELLOW

Question 6

describe BLOOD AGAR PLATES

Answer 6

• are STANDARD NUTRIENT PLATES that have an added 5% SHEEP BLOOD ADDED
• type of DIFFERENTIAL MEDIA; looking for THREE DIFFERENT TYPES OF HEMOLYSIS

CONDITIONS:

• BETA-HEMOLYTIC: organisms that PRODUCE ENZYME HEMOLYSIN – has CLEARING AROUND COLONIES/COMPLETE DIGESTION of RBCs
• ALPHA-HEMOLYTIC: organisms that PRODUCE ENZYMES that only PARTIALLY DIGEST THE RBCS– often seen in GREENISH-COLORED GROWTH
• GAMMA-HEMOLYTIC: has NO HEMOLYSIS/NO HEMOLYSIN PRODUCED

Question 7

describe the OXIDATION-FERMENTATION (O-F) TEST

Answer 7

• test used to DETERMINE if an ORGANISM’s CARBOHYDRATE METABOLISM is OXIDATIVE or FERMENTATIVE

CONDITIONS:

• use of TWO TUBES of LIFSON’S OF BASAL MEDIA
• has pH INDICATOR of BROMOTHYMOL BLUE
• use of MINERAL OIL on ONE TUBE; creation of ANAEROBIC ENVIRONMENT
• OXIDATIVE; AEROBIC
• FERMENTATION; ANAEROBIC

RESULTS:

• BOTH YELLOW; = (+) FERMENTATION
• YELLOW (OPEN), GREEN (SEALED) = (+) OXIDATION
• BOTH GREEN; NO METABOLISM of CARB/SUGAR

why?
- OXYGEN UTILIZED in OXIDATION

Question 8

describe FERMENTATION of CARBS/PHENOL RED FERMENTATION BROTH TEST

Answer 8

• test utilizes to test an organism’s ABILITY TO FERMENT CARBOHYDRATES
• FERMENTATION: the ANAEROBIC DEGRADATION of a SUBSTANCE (CARBS)
• END PRODUCTS: CO2 + ACIDS + ALCOHOL

MATERIALS:

• FERMENTATION BROTH; has ONE CARBOHYDRATE (typically GLUCOSE, SUCROSE, LACTOSE, and MANNITOL–used GLUCOSE and LACTOSE in LAB)
• pH INDICATOR; PHENOL RED
• can also use a SMALL DURHAM TUBE to observe for GAS PRODUCTION

RESULTS:

• addition of PHENOL RED INDICATOR–
  CONDITIONS;

RED - pH 7
YELLOW - ACIDIC pH

** YELLOW: PRODUCTION OF ACIDS – INDICATORS FERMENTATION OF CARBOHYDRATE (+)
**RED: NO CARBOHYDRATE FERMENTATION–use of PROTEIN IN MEDIA (PEPTONE)–the creation of a more ALKALINE MEDIA

Question 9

describe the METHYL RED and VOGES-PROSKAUER TEST (MR/VP TEST)

Answer 9

• allows to look for END PRODUCTS of GLUCOSE METABOLISM
• helps to DISTINGUISH between MEMBERS of the LARGE ENTEROBACTERIACEAE FAMILY (E. coli + E. aerogenes)

RESULTS:
(+) MR RESULT; indication of PRODUCTION OF LARGE AMTS OF ACID from GLUCOSE METABOLISM (RED +, ORANGE/YELLOW -)
(+) VP RESULT; indicates the PRODUCTION of NEUTRAL SUBSTANCES such as ACETOIN or ALCOHOL (RED +, ORANGE/YELLOW -)

Question 10

definition of CARDINAL TEMPERATURES

Answer 10

• the species’ characterization and range of MINIMUM, MAXIMUM, and OPTIMUM TEMPERATURES
• baseline of temp of lowest and highest for growth
• OPTIMUM; temp at which the ORGANISM GROWS THE FASTEST

Question 11

what are PSYCHROPHILES?

Answer 11

organisms that ONLY GROW below 20 degrees Celsius

• often seen in the OCEAN or ANTARTICA

Question 12

what are PSYCHROTROPHS?

Answer 12

organisms that have ADAPTED to COLD HABITATS, can FLUCTATE from around 0 - 30 Celsius

Question 13

what are MESOPHILES?

Answer 13

organisms that have ADAPTED to TEMPERATURES around 15 - 45 Celsius

Question 14

what are THERMOPHILES?

Answer 14

organisms that are ADAPTED to TEMPERATURES ABOVE 40 Celsius

Question 15

what are OBLIGATE THERMOPHILES and FACULTATIVE THERMOPHILES?

Answer 15

OBLIGATE THERMOPHILES:

• CANNOT GROW BELOW 40 C

FACULTATIVE THERMOPHILES:

• CAN GROW BELOW 40 C

Question 16

what are EXTREME THERMOPHILES?

Answer 16

organisms that survive in temperatures ranging from 65 C - 110 C

Question 17

definition of pH

Answer 17

the CONVENTIONAL MEANS of EXPRESSING the CONCENTRATION/ACTIVITY of HYDROGEN IONS in a SOLUTION

Question 18

what are ACIDOPHILES?

Answer 18

organisms that grow well in environments that are BELOW pH 5.5

Question 19

what are NEUTROPHILES?

Answer 19

organisms that prefer pH levels around 5.5 - 8.5

Question 20

what are ALKALIPHILES?

Answer 20

organisms that LIVE ABOVE pH 8.5

Question 21

what happens if an organisms MOVES OUT OF their typical INTERNAL ENVIRONMENT?

Answer 21

• can DESTROY NECESSARY MEMBRANE POTENTIAL
• have DENATURING of VITAL ENZYMES

Question 22

describe CITRATE UTILIZATION TEST

Answer 22

• determines the ABILITY of an ORGANISM to UTILIZE CITRATE as a CARBON SOURCE
• use of SODIUM CITRATE & AMMONIUM PHOSPHATE > AMMONIA & AMMONIUM HYDROXIDE
• can help to DIFFERENTIATE between E. coli and Enterobacter

CONDITIONS:

• inoculation of SIMMONS CITRATE SLANTS with SWABS (used q-tips :) )
• use of pH INDICATOR BROMOTHYMOL BLUE

RESULTS:

GREEN (NEUTRAL pH) - NEGATIVE
BLUE (ALKALINE) - POSITIVE **Citrate was used

Question 23

describe the OXIDASE TEST

Answer 23

• test used to IDENTIFY THE PRESENCE of the OXIDASE ENZYME > (able to OXIDIZE) CYTOCHROME C –specific ELECTRON ACCEPTOR IN THE ELECTRON TRANSFER SYSTEM
• used to DIFFERENTIATE members of the PSEUDOMONADACEAE FAMILY (+) vs. the ENTEROBACTERIACEAE FAMILY ( - )

CONDITIONS:

• use of an OXIDASE STRIP and some WATER

RESULTS:

• POSITIVE RESULT; has DARK BLUE COLOR
• NEGATIVE RESULT; has NO COLOR

Question 24

why is it IMPORTANT to identify OXYGEN as an ULTIMATE ELECTRON ACCEPTOR?

Answer 24

• can result in a TOXIC OXYGEN REACTIVE SPECIES

OBLLIGATE AEROBES: + CATALASE
FACULTATIVE ANAEROBES: + CATALASE
AEROTOLERANT ANAEROBES: - CATALASE
OBLIGATE ANAEROBES: - CATALASE
MICROAEROPHILES: +/- CATALASE

Question 25

describe the CATALASE TEST

Answer 25

• test looks for the PRESENCE OF CATALASE–specific ENZYME PRESENT in the CYTOCHROME that contains AEROBIC & FACULTATIVE ANAEROBES

RESULTS:
- NEGATIVE; NO BUBBLING
- POSITIVE; BUBBLING

*also helps to DIFFERENTIATE between GENERA;
- STREPTOCOCCUS (-) from STAPHYLOCOCCUS
- MICROCOCCUS (+) BACILLUS from CLOSTRIDIUM (-)

Question 26

describe the DECARBOXYLATION of AMINO ACIDS

Answer 26

• looking for the ABILITY TO DECARBOXYLATE AMINO ACIDS
• DIFFERENTIATES the MEMBERS of the ENTEROBACTERIACEAE FAMILY

CONDITIONS:

• use of DECARBOYXLATION BROTH that has ONE AMINO ACID (LYSINE, ARGININE, or ORNITHINE)
• use of pH INDICATOR BROMOCRESOL PURPLE
  (is PURPLE in ALKALINE CONDITIONS)

RESULTS:

(+) RESULT; remains PURPLE/DARK PURPLE - AA was DECARBOXYLATED

( - ) RESULT; turns YELLOW or CLEAR due to ACID PRODUCTION

Question 27

describe the STARCH HYDROLYSIS TEST

Answer 27

• detects the PRODUCTION of the EXOENZYME AMYLASE
• enzyme DEGRADES STARCH (POLYSACCHARIDE)&raquo_space; degraded more into MALTOSE (DISACCHARIDE) - taken up by bacterial cells

CONDITIONS:

• use of STARCH AGAR
• use of IODINE SOLUTION
  **INOCULATE SA PLATE + FLOOD WITH IODINE

RESULTS:

POSITIVE RESULT: CLEARING
NEGATIVE RESULT: PLATE will REMAIN BLUE/BLACK

Question 28

describe the UREASE TEST

Answer 28

• used to determine the ABILITY of an ORGANISM to USE UREA as a SOURCE OF NITROGEN – by UTILIZING UREASE
• DIFFERENTIATES the PROTEUS SPECIES and IDENTIFIES Helicobacter pylori

RESULTS:

• PHENOL RED INDICATOR;

media turns more ALKALINE–changing of COLOR

POSITIVE UREA TEST: PINK/RED
NEGATIVE UREA TEST: YELLOW/LIGHT ORANGE

Question 29

describe the SIM TEST

Answer 29

• helps to determine SULFUR REDUCTION + INDOLE PRODUCTION + MOTILITY

SULFUR REDUCTION;

• media contains IRON SALT – interacts with H2S; forms a BLACK PRECIPITATE
• POSITIVE; a BLACK PRECIPITATE – due to BREAK DOWN of AA CYSTENINE (sulfur-containing AA) to HYDROGEN SULFIDE OR has ENZYME THIOSULFATE REDUCTASE – reduces sulfur to H2S
• NEGATIVE; stays CLEAR

INDOLE REDUCTION:

• contains TRYPTOPHAN; looks for BREAKDOWN of AA TRYPTOPHAN (with enzyme TRYPTOPHANASE) > INDOLE + AMMONIA + PYRUVIC ACID
• addition of *KOVAC

POSITIVE RESULTS: RED
NEGATIVE RESULTS; YELLOW

MOTILITY:

• medium is SEMISOLID
• POSITIVE RESULTS; HAZY + MOVES AWAY FROM STAB INOCULATION
• NEGATIVE; stays close to STAB

*allows to ALSO DIFFERENTIATE the MEMBERS of the ENTEROBACTERIACEAE FAMILY