1. Week 8 W New: Exp 24: IMViC Part A: Indole Test Part B: Citrate Test
Exp 25: Hydrogen Sulfide Test
Exp 26: Urease Test
Exp 30: Oxidase Test
2nd
Exp 22: Carbohydrate Fermentation Lab Two pg 158
Exp 23: Triple Sugar Iron Agar Test Lab Two pg 162
Charts
1st
10/10

2. Lactose Fermentation—which one is positive?
Fermentation of glucose, lactose and sucrose – In this set of tests, you will be able to determine if the bacterium can ferment glucose, can hydrolyze lactose (into glucose and galactose and then ferment either of the monomers released, usually only the glucose), and can hydrolyze sucrose (into glucose and fructose and then ferment either of the monomers released). Fermentation simply uses an organic molecule as an electron acceptor, with the result being the production of organic acids (and a pH change in the medium). You will also be able to determine if the bacterium can produce a gas (usually CO2) during the fermentation process.

3. Ex 22: Carbohydrate Fermentation (by facultative anerobes)
Fermentation w/ Phenol Red (pH indicator)
- Red at neutral ph (7)= no Fermentaion occured
A Yellow at acidic ph (<7)= Fermentation
G microorganisms that produce gas (CO2) will
show as a bubble in the inverted Durham tube
Some bacteria metabolize proteins (peptones) for energy instead of the carbohydrate, which cause the broth to turn basic (alkaline).
Facultative anaerobes (often callled the fermenters of carbo.) are able to degrade glucose by the glycolytic pathway to produce pyruvic acid and then organic acids (lactic, formic,succinic acid or acetic acid) and gas (CO2 and H2) to produce ATP. If the solution has turned yellow (procution of acids lowering pH) w/ or w/o gas evolultion in Durham tube then pos rxn.

4. Results To ID Gram Negative Bacteria Serratia marcescens (SM)
Notation
MICROORGANISM
E.coli (EC)
P. aeruginosa (PA)
P.vulgaris (PV)
E. aerogenes (EA)
Serratia marcescens (SM)
Morphology
shape/ arrangement
rods
TSA (cultural characteristics)
color, size, form, margin, elevation, texture
white, small, circular, entire, convex , wet
white/green, small, circular, entire, convex, mucoid
white, small, circular, undulate, wet
white, small, circular, entire, convex, wet
red centers, small, circular, entire, convex, wet
PEA
+/- growth
MSA
Starch Hydrolysis
+/- amylase
Gelatin Liquefac.
+/- gelatinase ■
Lactose Ferm.
A (G) /- ferm.
Dextrose Ferm. (glucose)
Sucrose Ferm.
TSI Slant
Alk or A
TSI Butt
Alk or A (G)
TSI H2S
+/- H2S prod.
SIM Indole Prod.
+/- tryptophanase
SIM H2S
SIM Motility
+/- motility
Citrate Ferm.
+/- ferm.
Urease Activity
+/- urease
Methyl Red
+/-
VP (Voges-Proskauer)
Oxidase Activity
+/- oxidase
Catalase Activity
+/- catalase
NO3 (nitrate reduction)
Coagulase Actvity
+/- coagulase
+ - positive
AG - acid with gas
MF - mannitol fermentor
- - negative
± - variable
LF - lactose fermentor
Lucia Testa: Bio 156
Members of the Enterobacteriaceae are rod-shaped, and are typically 1-5 μm in length, have Gram-negative stains,and they are facultative anaerobes, fermenting sugars to produce lactic acid and various other end products. Most also reduce nitrate to nitrite. Unlike most similar bacteria, Enterobacteriaceae generally lack cytochrome C oxidase. Most have many flagella used to move about, but a few genera are non-motile. Catalase reactions vary among Enterobacteriaceae.
Many members of this family are a normal part of the gut flora found in the intestines of humans and other animals, while others are found in water or soil, or are parasites on a variety of different animals and plants. Escherichia coli, better known as E. coli, is one of the most important model organisms, and its genetics and biochemistry have been closely studied.
Pseudomonas aeruginosa is a common bacterium which can cause disease in animals and humans. It is found in soil, water, skin flora and most man-made environments throughout the world. It thrives not only in normal atmospheres, but also with little oxygen, and has thus colonised many natural and artificial environments. It uses a wide range of organic material for food; in animals, the versatility enables the organism to infect damaged tissues or people with reduced immunity. The symptoms of such infections are generalised inflammation and sepsis. Because it thrives on most surfaces, this bacterium is also found on and in medical equipment including catheters, causing cross infections in hospitals and clinics. It is implicated in hot-tub rash.
It is a Gram-negative, aerobic, rod-shaped bacterium with unipolar motility.[2] An opportunistic human pathogen, P. aeruginosa is also an opportunistic pathogen of plants.P. aeruginosa secretes a variety of pigments, including pyocyanin (blue-green), fluorescein (yellow-green )P. aeruginosa is often preliminarily identified by its pearlescent appearance and grape-like or tortilla-like odour in vitro.
Although classified as an aerobic organism, P. aeruginosa is considered by many as a facultative anaerobe, as it is well adapted to proliferate in conditions of partial or total oxygen depletion. This organism can achieve anaerobic growth with nitrate as a terminal electron acceptor, Adaptation to microaerobic or anaerobic environments is essential for certain lifestyles of P. aeruginosa, for example, during lung infection in cystic fibrosis patients, where thick layers of alginate surrounding bacterial mucoid cells can limit the diffusion of oxygen.

5. TSI Results
TSI distinguishes between Enterobacteriaceae and other types of intestinal bacilli
All Enterobacteriaceae ferment glucose and produce acid
TSI distinguishes among members of Enterobacteriaceae
In carbohydrate fermentation patterns
In whether or not they produce hydrogen sulfide (H2S)

6. Exp. 23:TRIPLE SUGAR IRON AGAR
COLOR/CONDITION
INTERPRETATION
RED SLANT/ RED BUTT
(Alk/Alk)
No carbohydrate fermentation Instead, peptones are catabolized aerobically and/or anaerobically.
RED SLANT/ YELLOW BUTT
(Alk/A)
Only glucose fermentation has occurred. Peptones are catabolized aerobically.
YELLOW SLANT/ YELLOW BUTT
(A/A)
Lactose and/or sucrose fermentation has occurred.
GAS FORMED (G)
Seen as cracks in the agar, bubbles, or the entire slant may be pushed out of the tube.
BLACK PRECIPITATE (+/-)
H2S has been produced.
Alk Slant/ A Butt w/ or w/o gas prod- Only glu. ferm. has occurred . Alk Slant Just a little acid was prod from glu ferm (glu in minimal conc) so got oxidiaed(alk), then alk prod from peptone degradation. Showed red. A Butt Acid maintained b/c ↓ O2 and slower growth of orgs.
A Slant/ A Butt w/ or w/o gas prod- Lactose and/or sucrose ferm. has occurred. Since both lac and suc are present in ↑ conc, they are continual subst. to ferm in both slant and butt.
Alk Slant/ Alk Butt- No carbo ferm has occurred. Peptones are catabolizes under aerobic (slant) and anerobic (butt) conditions. If Alk Slant/ no change Butt (orange-red), then only aerobic degradation occurred.
Sodium Thiosulfate subst. for H2S prod.(colorless) Orgs capable of H2S prod. will show blackening of butt b/c of ppt of insol ferrous sulfide.

7. How to Identify or Differentiate an Organism (based on what we have learned to date)
Then use reference sources: Bergey’s Manual, Lab Manual & Journal

8. Ex 24: IMViC Part A: Indole Test
IMViC Enterobacteriaeae (enterics) are Gram-negative bacteria that grow in the intestinal tract of humans and other animals. The IMViC tests are frequently employed for identification of this group of microbes which includes such organisms as Klebsiella, Enterobacter, and Escherichia coli. The presence of E. coli is used by public health officials as an indicator of fecal contamination of food and water supplies. While Enterobacter and Klebsiella resemble E.coli in being lactose fermenters, their presence does not necessarily indicate fecal contamination because they are widespread in soil and grass. The IMViC tests can be used to differentiate these three organisms.
IMViC is an acronym that stands for indole , methyl red, Voges-Proskauer , and citrate . To obtain the results of these four tests, three test tubes are inoculated: tryptone broth (indole test), methyl red - Voges Proskauer broth (MR-VP broth), and citrate.
The Indole Test
The test organism is inoculated into tryptone broth, a rich source of the amino acid tryptophan. Indole positive bacteria such as Escherichia coli produce tryptophanase, an enzyme that cleaves tryptophan, producing indole and other products. When Kovac's reagent (p-dimethylaminobenzaldehyde) is added to a broth with indole in it, a dark pink color develops. The indole test must be read by 48 hours of incubation because the indole can be further degraded if prolonged incubation occurs. The acidic pH produced by Escherichia coli limits its growth.
Used to distinguish between microorganisms that can break down the amino acid tryptophan
Tryptophan is an essential amino acid; some organisms possess an enzyme, tryptophanase, that can metabolize it
Indole is a product of tryptophan metabolism
SIM agar contains tryptophan
Kovac’s reagent reacts with the indole and produces a cherry red layer on the top of the media—the presence of the cherry red layer means that the organism can break down tryptophan (indole-positive reaction)

9. Medium: SIM Deep
Chemical Components:
Tryptophan and Kovac’s Reagent (indicator)
Property/Enzyme Testing For: Tryptohanase degrades tryptophan to prod indole
Results:
+ = cherry red layer - = no color change
Interpretation of Results: Kovac’s Reagent tests for indole- a byproduct of tryptophan degradation. Absence of red coloration demonstrates that the substrate tryptophan was not hydrolyzed and indicates an indole- neg reaction due to lack of tryptophanase.

10. Ex 25: Hydrogen Sulfide Test (& motility)
Used to determine which organisms produce H2S gas by reducing sulfur-containing compounds present in the medium
The SIM medium contains peptones and sodium thiosulfate.
Gaseous H2S may be produced in two ways by bacteria.  SIM agar may be used to detect this production of gas.  SIM agar contains peptone, sodium thiosulfate, and ferrous ammonium sulfate.  When hydrogen sulfide gas is produced, a precipitation reaction will occur with the ferrous ammonium sulfate.  An insoluble black precipitate is seen as a positive result.
  Hydrogen Sulfide Test on SIM
The tube on the left is a negative result and the tube on the right is positive.
Peptones are broken down to their constituent amino acids; cysteine is a sulfur-containing amino acid
Some microorganisms have an enzyme that metabolizes cysteine, with H2S gas as a product
Some microorganisms can reduce the S2O32- to sulfite, with H2S as a product
Ferrous sulfate is an indicator; it combines with the gas and turns black in the medium
Pathway1: Some bacteria can degrade peptones to aa-- including sulfer-containing aa (such as cysteine- source of H2S prod.). Pathway 2: Inorgainc sodium thiosulfate subst. for H2S prod. Orgs capable of H2S (colorless) prod. will show blackening of butt b/c of ppt of insol ferrous sulfide. This is a differential medium. It tests the ability of an organism to do several things: reduce sulfur, produce indole and swim through the agar (be motile). SIM is commonly used to differentiate members of Enterobacteriaceae. Sulfur can be reduced to H2S (hydrogen sulfide) either by catabolism of the amino acid cysteine by the enzyme cysteine desulfurase or by reduction of thiosulfate in anaerobic respiration. If hydrogen sulfide is produced, a black color forms in the medium. Proteus mirabilis is positive for H2S production. The organism pictured on the far left is positive for hydrogen sulfide production. Bacteria that have the enzyme tryptophanase, can convert the amino acid, tryptophane to indole. Indole reacts with added Kovac’s reagent to form rosindole dye which is red in color (indole +). Escherichia coli is indole positive. The organism pictured second from left is indole positive. SIM tubes are inoculated with a single stab to the bottom of the tube. If an organism is motile than the growth will radiate from the stab mark and make the entire tube appear turbid. Pseudomonas aeruginosa and the strain of Proteus mirabilis that we work with are motile

11. Medium: SIM Deep
Chemical Components:
Cysteine and Thiosulfate Ferrous sulfate (indicator)
Property/Enzyme Testing For: Ability to ferment sulfur-containing amino acids or inorganic sulfur compounds
Results:
+ = black precipitate - = no black precipitate
Interpretation of Results: Organisms capable of H2S (colorless) production, will show blackening of the butt b/c of precipitation of insoluble ferrous sulfide. [Pathway 1: Some bacteria can degrade sulfur-containing peptones to amino acids (such as cysteine- source of H2S). Pathway 2: Inorgainc sodium thiosulfate is the substrate for H2S. ]

12. Medium: SIM Deep
Chemical Components:
not applicable
Property/Enzyme Testing For: flagellated organisms
Results:
+ = motile - = nonmotile
Interpretation of Results: When culture growth (turbidity) of flagellated organisms is not restricted to the line of inoculation.

13. Ex 24: IMViC Part D: Citrate Test
The Citrate Test
The citrate test utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source. Simmon's media contains bromthymol blue, a pH indicator with a range of 6.0 to 7.6. Bromthymol blue is yellow at acidic pH's (around 6), and gradually changes to blue at more alkaline pH's (around 7.6). Uninoculated Simmon's citrate agar has a pH of 6.9, so it is an intermediate green color. Growth of bacteria in the media leads to development of a Prussian blue color (positive citrate). Enterobacter and Klebsiella are citrate positive while E.coli is negative.
Thus E.coli gives ++-- results on the IMViC tests, while Enterobacter and Klebsiella give the reverse: --++
Used to determine which microorganisms can use the molecule citrate as the only carbon source to ferment
Those that can break down citrate to produce energy contain the enzyme citrate permease that transports citrate into the cell; citrate is metabolized using the enzyme citrase which occurs in the Krebs cycle
Citrate is broken down into pyruvic and acetic acids, with CO2 reacting with H2O and Na+ to produce sodium bicarbonate as a byproduct. This is basic and will turn a special indicator blue
In absense of ferm glu or lac, some bacteria capable to ferm citrate. Byprod of this rxn are alk (emission of CO2 which combines w/salt to form Na2CO3) which ∆ Bromthymol Blue from green to blue.
Bottom line: those organisms that ferment citrate will show growth on the slant and blue coloration; lack of citrate fermentation will cause no growth and medium will remain green

14. Medium: Citrate Agar Slant
Chemical Components:
Bromthymol blue (indicator)
Property/Enzyme Testing For: Capability of using citrate as a carbon source.
Results:
+ = blue - = green
Interpretation of Results: In absence of fermentable glucose or lactose, some bacteria are capable of fermenting citrate. Byproducts of this reaction are alkaline (emission of CO2 combines with salt to form Na2CO3) which changes bromthymol blue from green to blue.

15. Ex 26: Urease Test
Used to determine if a microorganism can degrade urea
Key identifier for Proteus vulgaris, which has the enzyme urease
Urease is a hydrolytic enzyme that breaks down urea and produces CO2, H2O, and ammonia
Phenol red (pH indicator) is present; it turns yellow in acidic conditions and red in basic conditions
Organisms that possess urease will turn the broth red (ammonia is a product and it is basic!)
Esp helpful to id Proteus vulgaris. As orgs splits urea, presence of alk ammonia (NH3) creates alk environ turning phenol red deep pink.

16. Medium: Urea broth
Chemical Components:
Phenol red (indicator)
Property/Enzyme Testing For: Urease degrades amide compounds.
Results:
+ = bright pink - = salmon color (no change)
Interpretation of Results: Especially helpful to identify Proteus vulgaris. As organisms splits urea, presence of alkaline ammonia (NH3) creates alkaline environment turning phenol red deep pink.

17. Exp. 30: Oxidase Test
Used to determine which organisms utilize the enzyme cytochrome oxidase
Cytochrome oxidase is part of the electron transport chain and transfers electrons from a donor molecule to oxygen
Aerobic bacteria, and some facultative anaerobes, exhibit oxidase activity
Pseudomonas is oxidase positive
Enterobacteriaceae are oxidase negative
A test reagent (p-aminodimethylaniline oxalate) changes color in the presence of oxidase
Maroon-black = positive for oxidase
Psudamonas auruginosa and aerobic, some facultative anerobes, and microaerophiles are oxd pos whereas Enterobacteriaceae (E. coli and Enterobacter aurugenes) oxd neg. Oxidase imp in ETC during aerobic resp. Oxalate reagent serves as a e- donor becoming oxidized to a blackish comp in the presence of ozidase and free O2.
No change in color = negative

18. Medium: TSA plate
Chemical Components:
Oxalate
Property/Enzyme Testing For: Cytochrome oxidase aids aerobic respiration
Results:
+ = purple/black - = no color change or light pink
Interpretation of Results: Pseudomonas auruginosa, all aerobic, some facultative anerobes, and microaerophiles are oxidative positive. Whereas Enterobacteriaceae (E. coli and Enterobacter aurugenes) are oxidative negative. Oxidase is important in the electron transfer chain during aerobic respiration. Oxalate reagent serves as an e- donor becoming oxidized to a blackish color in the presence of oxidase and free O2.

19. WEEK 8w TESTS (per table) CULTURE MEDIA PROCEDURE Ex 24: IMViC
Part A: Indole Test
Ex 25: Hydrogen
Sulfide Test
(& motility)
EC, PA, PV, EA
4 SIM deeps
Ex 24 pt A: Ref. pages:
Ex 25: Ref. pages:178.
Stab SIM in center to butt w/ needle.
- Incubation: 37oC 24 Hrs.
- Next week: add Kovac’s reagent for
indole test.
Part D: Citrate Test
4 Citrate slants
- Ref. pages:
- Streak surface w/ needle.
Ex 26: Urease Test
4 Urea broths
Ref. pages:
- Heavily inoculate: use 2 loopfulls of org.
Ex 30: Oxidase Test
2 TSA plates
(2organism
/plate)
Ref. pages:
Divide plates in half, single line steak org.
Incubation: 37oC 24 Hrs.
Next week: add oxalate.