what is added to the sim media so the black precipitate will form indicating h2s production?

Lab 11: Biochemical Tests (Mean solar day 2)

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Assistant Professor (Biological Sciences) at California Land Academy

BIOCHEMICAL TESTS (Solar day two):

catalase exam

Tests for the presence of catalase enzyme that converts hydrogen peroxide (H₂O₂) into h2o and oxygen gas

oxidase test

Used to determine if a bacterium has enzyme cytochrome oxidase. The reagent is colorless if negative and turns blue/purple when oxidized.

CATALASE TEST:

1. On a glass slide, add together 1-2 drops of H₂O₂.

2. Add loopful of a single species of bacteria from your plate to the slide; bubbles = positive result

iii. Repeat for all iv bacteria AND YOUR ASSIGNED Bacteria from your TSA plate

OXIDASE Examination:

iv. Swipe bacteria from your assigned PLATE onto a cotton swab.

5. Manipulate i-2 drops of the oxidase reagent onto the swab and record result.

half-dozen. Repeat for all four bacteria AND YOUR ASSIGNED Leaner from your TSA plate.

Bacteria	Catalase +/-	Oxidase +/-
	+	+
	+	-
	+	-
	-	-

NITRATE Broth:

Complete the post-obit for each tube of the 4 tubes of nitrate broth. Observe the inverted Durham tube. If bubbles are nowadays, then the following reaction has occurred and your bacteria is positive for nitrite and nitrate reductase:

NO₃ (nitrate) → NO₂ (nitrite) → N₂ (gas)

1. If gas is NOT present, and then add together 3 drops of sufanilic acrid (reagent A) and 3 drops of naphthlamine (reagent B) to your tube. Mix gently by tapping the bottom of the tube and wait 2-3 minutes. If your tube turns cerise, and then bacteria is positive for nitrate reductase and the following reaction has occurred:

NO₃ → NO₂

two. If no red color is observed, add a TINY amount of powdered zinc using a toothpick and mix gently past tapping the lesser of the tube.

Notation

If your sample turns red subsequently footstep ii, Do NOT exercise step 3

3. Wait 15 minutes. If your tube turns red, then NO₃ is still nowadays and leaner are nitrate reductase negative.

Note

Hint for lab practical – if I requite y'all a red tube, how tin you lot tell if it is nitrate positive or negative?

	NO_three present in the tube?	NO_ii present in the tube?	Due north_ii gas nowadays in the tube?
Assigned leaner
Alcaligenes faecalis
Escherichia coli
Pseudomonas aeruginosa
	NO₃ is detected by?	NO₂ is detected by?	Due north₂ is detected by?

STARCH PLATES:

4. Add enough iodine reagent to flood your plate. Await 5 MINUTES. The presence of clear halos surrounding colonies is positive for their ability to digest the starch and indicates the presence of alpha-amylase.

GELATINASE Examination:

5. Place your gelatin tube in the water ice bucket provided past the instructor for 10 minutes. So, hold your tube sideways to determine if it is solid (negative result) or liquid (positive result). RECORD RESULTS FOR:

CASEIN plate
TRIBUTYRIN plate
Phenol Cerise Broths

TSI SLANTS:

half-dozen. Record your results for your assigned bacteria using the tabular array beneath.

RESULTS (slant/butt)	SYMBOL	INTERPRETATION
Red/yellow	M/A	Glucose fermentation only; Peptone catabolized.
Yellowish/yellow	A/A	Glucose and lactose and/or sucrose fermentation
Blood-red/red	K/K	No fermentation; Peptone catabolized.
Red/no color change	Chiliad/NC	No fermentation; Peptone used aerobically
Yellowish/yellow with bubbling	A/A,G	Glucose and lactose and/or sucrose fermentation; Gas produced.
Red/yellow with bubbles	Chiliad/A,G	Glucose fermentation only; Gas produced.
Red/yellow with bubbles and black precipitate	K/A,G,H_iiS	Glucose fermentation simply; Gas produced; H_twoS produced.
Cerise/xanthous with black precipitate	K/A,H₂S	Glucose fermentation only; H₂S produced.
Xanthous/yellow with black precipitate	A/A, H₂S	Glucose and lactose and/or sucrose fermentation; H2S produced.
No change/no change	NC/NC	No fermentation
A=acrid product; Chiliad=gas production, K=alkaline metal reaction; H₂S=sulfur reduction

SIM MEDIA:

7. Add together 5 drops of Kovacs reagent to the tube to detect indole production.

Sulfur	-	+	+	-
Indole	+	-	-	-
Movement	-	+	-	-

MR-VP:

eight. Using a drinking glass Pasteur pipet, take ane ml out of your MR-VP broth tube and identify information technology into a new tube

9. Add together 5 drops MR methyl ruddy reagent to the new tube, record results (positive outcome = red)

10. Using a glass pipet accept 1 ml out of MR-VP broth tube and place it into a new tube

11. Add half-dozen drops VP-A and three drops VP-B, record results (positive result = red)

RECORD RESULTS FOR:

Decarboxylate tubes
Urea broth
MAC plate

INDEX:

CASEIN (MILK) PLATE:

Casein is a large globular protein that gives milk its white and opaque color. It is too large to exist transported beyond the jail cell membrane. Bacteria that have the exoenzyme 'casease' are able to hydrolyze casein by secreting this enzyme into the environment effectually them. Articulate halos surrounding colonies are indicative of their ability to digest the casein and results in a zone of clearance around plated growth. (+) = halo (-) = no halo

SELECTIVE Amanuensis: none

DIFFERENTIAL Amanuensis: casein

INDICATOR: none

CATALASE Examination:

Catalases are enzymes that convert hydrogen peroxide (H2O2) into water and oxygen gas. When a drop of peroxide is placed on catalase-producing bacteria, bubbles announced when the oxygen gas is formed. This is a test for aerobic (able to utilize oxygen) catalase-positive leaner such as Staphylococcus and Micrococcus.

CITRATE TEST:

Used for the detection of gram-negative enteric bacilli based on the power of an organism to use citrate as the sole source of carbon and free energy. Organisms capable of utilizing ammonium dihydrogen phosphate and citrate volition grow unrestricted on this medium. Bromothymol blue acts equally a pH indicator, causing the medium to change from light-green (neutral) to blueish (alkaline) with increasing pH. Citrate utilization produces an alkali metal carbonate, resulting in a deep blue color change within the agar. The medium will remain green if organisms practise not grow or are not able to metabolize sodium citrate.

SELECTIVE Amanuensis: none

DIFFERENTIAL AGENT: ammonium dihydrogen phosphate and sodium citrate

INDICATOR: bromothymol blue

DECARBOXYLATE BROTHS:

For the differentiation of gram-negative enteric bacilli based on their ability to break down specific amino acids. The production of the enzymes that intermission down amino acids (decarboxylase & dihydrolase enzymes) is induced in an acidic environment.

CONTROL: glucose fermented → acid → yellow

ARGININE, LYSINE or ORNITHINE: amino acrid degraded → basic → royal

Microorganisms possessing the specific decarboxylase and dihydrolase enzymes for the amino acrid (arginine, lysine or ornithine) dethrone the amino acid to yield various amine by-products which create an alkaline environment that turns the indicator purplish. If the organism does not produce the appropriate enzyme, and then the medium will remain xanthous.

DIFFERENTIAL AGENT: Arginine, lysine or ornithine amino acids

INDICATOR: bromocresol royal and cresol blood-red

GELATIN HYDROLYSIS TEST:

Nutrient gelatin is a differential medium that tests the power of an organism to produce an exoenzyme, chosen gelatinase, that hydrolyzes gelatin. When gelatin is cleaved downward, information technology can no longer solidify. If an organism tin break down gelatin, the areas where the organism has grown will remain liquid even if the gelatin is chilled.

Methyl Red and Voges-Proskauer (MR-VP):

A medium that contains protein, glucose, and phosphate buffer. Some bacteria produce an acid while other leaner further metabolize the acrid to pH stable finish products (Glucose→Acrid→Stable End Product). The MR test is used to notice organisms capable of performing mixed acrid fermentation where:

Red=positive
Orange=inconclusive
Yellow=negative

The VP Test is designed for organisms who are able to ferment glucose to acids which are and so converted to acetoin and ii,iii butanediol. The addition of VP reagents oxidizes acetoin to diacetyl and reacts with guanidine nuclei from the peptones to produce a red color. Remember:

Ruby=positive results
No color change or copper color=negative

MANNITOL SALT AGAR PLATE (MSA):

Selective for gram-positive bacteria (e.m. Staphylococcus and Micrococcus). Mannitol fermentation by pathogenic staphylococci, such as South. aureus, is indicated past the media changing to yellow.

SELECTIVE Amanuensis: NaCl (salt)

DIFFERENTIAL Agent: mannitol sugar fermentation

INDICATOR: Phenol red

NITRATE (NO3) Broth:

Determines whether the microbe produces the enzymes nitrate reductase and nitrite reductase. The ii enzymes catalyze two reactions involved in converting starting chemical compound nitrate into finish product nitrogen gas.

After incubating the nitrate broth, sulfanilic acid (reagent A or 1) and α-naphthylamine (reagent B or two) are added. If a cherry-colored compound forms then nitrate reduction has occurred (NO3→NO2).

An inverted Durham tube tests for the presence of nitrogen gas (North₂).

If neither a ruby-red color or gas is observed, then, confirmation is necessary that nitrate (NO₃) remains in the broth. A Pocket-size improver of zinc dust will catechumen the nitrate to nitrite and course a red colour. This test reaction is considered negative for nitrate reduction.

Nitrate reductase (+) = ruby later on reagent A & B added
Nitrite and nitrate reductase (+) = bubbling in durham tube
Nitrate & nitrite reductase (-) = carmine after the addition of zinc

OXIDASE Exam:

Used to determine if a bacterium has enzyme cytochrome oxidase. The final phase of bacterial respiration involves a serial of membrane-embedded components collectively known as the electron ship chain. The concluding pace in the chain may involve the use of the enzyme cytochrome oxidase, which catalyzes the oxidation of cytochrome c while reducing oxygen to form h2o. The reagent is usually N,Due north,N′,Northward′-tetramethyl-p-phenylenediamine (TMPD) or Due north,N-dimethyl-pphenylenediamine (DMPD), which is also a redox indicator. The reagent is colorless if negative, the color turns blueish/purple when oxidized.

PHENOL RED Goop:

A general-purpose differential test medium typically used to differentiate gram-negative enteric bacteria. It contains peptone, phenol red (a pH indicator), one carbohydrate (i.e glucose or lactose), and a Durham tube to test for the ability to convert the terminate product of glycolysis, pyruvic acid, into CO2 gas.

DIFFERENTIAL AGENT: saccharide, Durham tube

INDICATOR: phenol red (-) (+)

SIM MEDIUM:

For the differentiation of gram-negative enteric bacilli on the basis of sulfide production, indole formation, and move. H2S production is detected when ferrous sulfide, a black precipitate, is produced as a result of ferrous ammonium sulfate reacting with H_twoS gas. Casein peptone is rich in tryptophan, bacteria that possess the enzyme tryptophanase degrade tryptophan to indole. Indole is detected upon the add-on of Kovacs Reagent producing a red band at the top of the medium. The semi-solid agar allows for the detection of bacterial move. Motile organisms extend from the stab line and produce turbidity or cloudiness throughout the medium. Non-motile organisms grow just along the stab line and go out the surrounding medium clear.

Sulfur	-	+	+	-
Indole	+	-	-	-
Motility	-	+	-	-

STARCH AGAR:

Starch is too big to laissez passer through the plasma membrane and must be split into private glucose molecules. Bacteria that have the exoenzyme amylase are able to hydrolyze starch by secreting these enzymes into the environment around them. Subsequently bacteria are allowed to grow, iodine is added to detect the presence of starch. Iodine complexes with starch to form a bluish-black color in the culture medium. Clear halos surrounding colonies is indicative of their ability to assimilate the starch and results in a zone of clearance effectually plated growth. (+) = halo (-) = no halo

SELECTIVE AGENTS: none

DIFFERENTIAL Agent: starch

INDICATOR: iodine

TRIBUTYRIN AGAR:

Tributyrin oil is a blazon of lipid called a triglyceride. It is too large to be transported beyond the cell membrane. Bacteria that have the exoenzyme lipase are able to hydrolyze tributyrin oil and break it down. Tributyrin oil forms an opaque interruption in the agar. When an organism produces lipase and breaks down the tributyrin, a clear halo surrounds the areas where the lipase-producing organism has grown. (+) = halo (-) = no halo

SELECTIVE AGENTS: none

DIFFERENTIAL AGENT: tributyrin oil

INDICATOR: none

TRIPLE Saccharide Fe (TSI) SLANT/DEEP:

Tests the power of bacteria to ferment sugars and to reduce sulfur to H₂S, often used to identify Salmonella and Shigella. Medium contains sugars (lactose, sucrose, and glucose) and thiosulfate. Slant/deep allows for aerobic and anaerobic growth conditions. An alkaline/acid (cherry-red camber/yellowish butt) reaction is indicative of glucose fermentation only. An acid/acid (yellow slant/yellow barrel) reaction indicates the fermentation of glucose, lactose and/or sucrose. The absenteeism of carbohydrate fermentation results in an alkaline/element of group i (reddish camber, red barrel) reaction. If an organism tin can reduce sulfur, the hydrogen sulfide (H₂S) gas which is produced will react with the ferrous ammonium sulfate ((NH₄)_twoFe(Then₄)₂ · 6H_twoO) to form fe sulfide (FeS), which appears equally a black precipitate. If CO_ii gas is produced, then the agar will be lifted or cracked as a result.

(A) Uninoculated

(B) 1000/K

(D) A/A+ H_twoS

Note

In (D), you tin't see the A because of the H₂S

DIFFERENTIAL AGENT: glucose, lactose, sucrose, and sodium thiosulfate

INDICATOR: phenol red and ferrous ammonium sulfate

UREA Goop:

Tests the power of an organism to hydrolyze urea to ammonia and carbon dioxide using the enzyme urease. The broth contains urea and a very small amount of nutrients for the bacteria. The pH indicator is phenol red. If the urea in the broth is degraded and ammonia is produced, the pH rises and the media turns pinkish.

BACTERIAL UNKNOWN IDENTIFICATION:

You must work independently on this projection! You volition receive no help in staining, performing tests, interpreting tests, or even focusing the microscope other than your lab notebook and your flowchart. There should be NO conversations betwixt students almost their unknown experiment. You Can Not take pictures. I reserve the right to penalize students for giving or receiving unauthorized assist from other students.

Each student will receive a broth culture of their unknown bacteria. The tube will be marked with a number merely. Record this number and write it on all plates and tubes that y'all inoculate, forth with your name and the date.

Your unknown sample will comprise i bacterial species.
Practise non discard any test results!
Yous must request the media that you need from the instructor do not take anything without request.

UNKNOWN LAB DAY ane:

Gram stain your culture and record the results on Data Sheet, and plow in your data canvas to the instructor (you lot will receive this document back at the get-go of twenty-four hours two).
Streak for isolated colonies on a TSA plate → this is a skills test.
Streak for isolated colonies on a MAC.
Store the original numbered unknown culture in your lab section's rack at the front of the class.

UNKNOWN LAB 24-hour interval 2:

Record the results of your MAC on Data Canvass #1.
Inoculate your chosen biochemical tests.
You get 3 groups of tests for free from the list below. After that, each examination will effect in the loss of five points.

EXO-ENZYMES (CASEIN AND TRYBUTERIN AND GEL)
DECARBOXYLATE (LYSINE AND ORNITHINE)
PHENOL RED BROTHS (GLUCOSE, LACTOSE, SUCROSE)
OXIDASE
TSI
NITRATE
UREA
CITRATE
MR-VP
SIM

UNKNOWN LAB Day 3:

Record your test results on your Information Canvass.
Identify the GENUS and SPECIES of your unknown bacteria.
Turn in Data SHEET.

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Source: https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/Book:_General_Microbiology_Lab_Manual_%28Pakpour_and_Horgan%29/Lab_11:_Biochemical_Tests_%28Day_2%29