Microbiology Lab Midterm

Question 1

Simple

Answer 1

Only one dye is used, crystal violet – cationic dye

Question 2

Negative stain

Answer 2

Nigrosin, black anionic dye, good for examining morphology and size of bacterial cells,NO HEAT

Question 3

Gram – differential stain detects peptidoglycan layer

Answer 3

• Crystal violet, positive Gram cells are blue
• Grahm’s iodine
• Ethanol
• Safranin-negative Gram cells are pink

Question 4

Acid-fast

Answer 4

(Ziehl–Neelsen method) differential stain – detects mycolic acid content in cell – Mycobacterium (tuberculosis and leprosy) and Nocardia

• Carbol fuchsin – steamed into cell, positive acid-fast cells are pink
• acid alcohol-decolorizer
• methylene blue-counterstain

Question 5

Endospore

Answer 5

(Schaeffer-Fulton method)

• Malachite green – steamed into organism, spores found in organism will be green, Bacillus, Clostridium, Sporosarcina all gram-positive spore forming bacteria) – structural stain
• water
• saffranin-pink-counterstain

Question 6

Phenylethyl alcohol– Medium

Answer 6

Selective – yes, inhibitor =phenylethyl alcohol, selects for = gram-positive, no differential, positive results = growth = staphylococci, negative results = no growth

Question 7

MacConkey Agar– Medium

Answer 7

Selective – yes, inhibitor = crystal violet and bile salts, selects for gram-negative, differential = yes, neutral red indicator, differentiates for lactose fermenters and non-fermenters, positive results = growth and red color produced = lactose fermentation, negative results = no growth and no red color produced

Question 8

Blood agar– Medium

Answer 8

Only differential, indicator = red blood cells, differentiates – hemolysis, Beta Alpha Gamma, positive results = yellow ring around colony, negative results = no hemolysis around colony

Question 9

Eosin methylene blue – Medium

Answer 9

Selective – yes, 
inhibitor = eosin and methylene blue, 
selects for gram-negative, 
differential – yes, 
indicator =eosin and methylene blue,
 differentiates = lactose fermenter, 
positive results = color change and growth (E. coli has green sheen, Enterobacter has pink), 
negative results = Staphylococcus no change

Question 10

Tetrazolium chloride– Medium

Answer 10

For motility
Only differential, indicator turns red if organism is present, positive result is redness throughout tube while negative result is redness were stab line is

Question 11

General purpose

Answer 11

support growth a variety of microorganisms

Question 12

Selective

Answer 12

use inhibitors to prevent growth of certain organisms

Question 13

Differential

Answer 13

allow growth of many microbes but differentiation is seen by indicators after change has occurred

Question 14

Combination

Answer 14

selective and differential

Question 15

What does a viable plate count do?

Answer 15

Method for diluting a broth culture so that a sample can be plated, viable cells are those that can replicate and form colonies

Question 16

What does viable plate count tell you?

Answer 16

Determine original concentration of the starting sample

Question 17

Countable range

Answer 17

30 – 300 CFU

Question 18

Litmus milk

Answer 18

Fermentation of lactose = pink,
intense fermentation = curding,
peptone deamination = blue,
redox reaction = white,
proteolysis=clear (bacteria has caseinase),
indicators – litmus pH, casein and lactose

Question 19

Sugar fermentation broth

Answer 19

Indicators = phenol red and Durham tube, yellow = acid from acid fermentation, base = cerise from peptone deamination, bubbles = gas

Question 20

Kliglers Iron agar 1% lactose, 0.1% glucose, 1% peptone-

Answer 20

Differentiates lactose fermentation, glucose fermentation, sulfur reduction

• indicators = phenol red and iron from ferric ammonium citrate which combines with H2S to form black precipitate
• lactose and glucose positive = entire tube yellow
• gaseous products form lifting and cracking
• glucose positive only bottom of tube is yellow top of tube is read from the emanation of peptones
• reversion from 2,3 butanediol pathway small amount of redness on top of tube but the organism is still glucose and lactose fermenter
• sulfur reduction = only glucose positive and black precipitate forms on bottom of tube

Question 21

What is the only H2S positive organism used

Answer 21

Proteos vulgaris

Question 22

Lipase – exoenzyme

Answer 22

Differential media, test for enzyme lipase, indicator = spirit blue dye, blue zone around bacterial colony

Question 23

Milk agar – exoenzyme

Answer 23

Differential
Test for caseinase, hydrolyzes casein into amino acids
-Milk is indicator
-Clear around colony positive result

Question 24

Starch agar – exoenzyme

Answer 24

Differential media
Test for the enzyme amylase, amylase hydrolyzes starch into simple sugars
-Colorless around colony = degradation of starch
-Iodine added

Question 25

Catalase test

Answer 25

Detects catalase, which converts hydrogen peroxide to water oxygen

• 3% hydrogen peroxide is added to the edge of the colony
• if bubbles are present than sample is positive

Question 26

Oxidase test

Answer 26

Detects for cytochrome C oxidase
-In the presence of cytochrome C oxidase reagent is oxidized and turns dark blue to black
-indicator=Dimethyl-p-phenylenediamine hydrochloride (an aromatic amine) is a reducing agent
FOR AEROBIC RESP

Question 27

Nitrate test

Answer 27

-If organism has nitrate reductase which converts nitrate to nitrite
-Product-Brick red color after nitrate one and nitrate to her added
-reagents for the added after incubation-Nitrate I-sulfanilic acid and Nitrate 2-dimethyl-alpha-naphthylamine…day 2=Zinc powder-positive result=Brick red color after nitrate one and nitrate 2 are added or absence of brick red when zinc is added
negative result=Brick red color after zinc is added

Question 28

Urea test

Answer 28

Is in broth medium
-detects urease which degrades urea into 2 ammonia and carbon dioxide
-cerise color is positive result due to increased alkalinity from breakdown
-yellow and red are negative results
there is a pH indicator present in the broth
** Proteus is urea positive organism

Question 29

Gelatin test

Answer 29

• Detects presence of gelatinase which hydrolyzes gelatin into amino acids
• After chilling in ice bath if broth is liquid the bacteria contains the enzyme
• solid at room temp=negative result

Question 30

Phenylalanine test

Answer 30

Medium detects production of phenylalanine deaminase which degrades phenylalanine into phenylpyruvic acid (PPA) and ammonia

• Deep green color president if bacteria contains enzyme
• Ferric chloride FeCl3 drops added
• PP in the pool turns green
• Yellow color bacteria does not contain enzyme
• Carried out on slant

Question 31

SIM test

Answer 31

S = sulfide, I = indole, and M= motility
Hydrogen sulfide=H2S– Detects presence of cysteine desulfurase which removes the sulfur sidechain from cysteine
Indole-from breakdown of tryptophan after 3-5 drops of Kovacs reagent = cerise color
motility = cloudiness

Remove sulfur forms a black precipitate in the presence of iron salts
-H2S plus iron = black

Cerise color = indole from tryptophan breakdown

Cloudiness = motility present

Question 32

Methyl red

Answer 32

Test for mixed acid fermenters, aftermath or red is added if reagent remains red then it is a mixed acid fermenter, only stays red in pH of 4.4 or less
-Methyl red reagent

Question 33

Voges-Proskauer

Answer 33

Test for 2,3-butanediol fermenter, these fermenters produce a acetoin intermediate,
VP1 +VP2+Acetoin=red
-If there is a red color then this is positive for a 2,3-butanediol fermenter
-VP 1-alpha naphtol
VP 2-KOH
Barritt’s reagant

Question 34

Tryptone broth

Answer 34

Detects tryptophanase enzyme which hydrolyzes tryptophan into indole, pyruvate and ammonia, cerise color = indole
-Kovacs reagent used, DMABA and HCl dissolved in amyl alcohol

Question 35

Simmons citrate

Answer 35

Detects for utilization of citrate as sole carbon source for growth, blue reaction may accompany growth but is not primary indicator growth is, Simmons is your bro
-bromothymol blue

Question 36

IMViC

Answer 36

Set of four tests that can be used to differentiate E. coli from Enterobacter

• E. coli = mixed acid while Enterobacter = butanediol pathway
• 4 tests used are Imdole, Methyl-Red,Voges-Proskauer,Citrate
• E. coli is positive for indole and methyl red negative for VP and citrate
• Enterobacter is positive for VP and citrate negative for indole and MR