Biochemical Tests PDF

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Biochemical tests, Microbiology ...

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Biochemical Test: Arginine Dihydrolase (Arginine Decarboxylase) Intended Use: To differentiate gram negative enteric bacilli based on the decarboxylation of arginine Principle: Fermentation of glucose establishes an acidic state, resulting in induction of arginine dihydrolase. Reagents/Media: Arginine Dihydrolase control (base) media, Arginine Dihydrolase test media, sterile mineral oil Positive Control: Enterobacter cloacae Negative Control: Klebsiella pneumoniae Step by Step instructions: 1.

Using 1-2 isolated colonies from a 18-24 hour culture, inoculate the test media

2.

Inoculate a control tube of dihydrolase base in parallel with the test media

3.

Overlay each inoculated tube with ~1 ml of sterile mineral oil

4.

Tighten the caps and incubate aerobically at 35C for 18-96 hours

5.

Observe daily for color reactions. Compare results to the inoculated control (base) media.

Test Interpretation: Positive

Initial fermentation of glucose results in a color shift to yellow; production of arginine dihydrolase will cause the media to revert back to its initial purple color

Negative

There is little to no color change, the media remains yellow

Biochemical Test: Bile Esculin

Intended Use: Selects and presumptively identifies Enterococcus spp. and Streptococcus bovis (Group D Strep). Used to identify non-hemolytic, catalase negative, gram positive cocci Principle: Differentiation is based on ability to hydrolyze esculin in the presence of 40% bile. Reagents/Media: Bile Esculin Agar Positive Control: Enterococcus faecalis Negative Control: Streptococcus agalactiae Step by Step instructions: 1.

Inoculate media with unknown sample by streaking the agar slant.

2.

Incubate culture at 35° Celsius.

3.

Observe culture after 24-48 hours.

Test Interpretation: Positive

Growth and Black pigmentation of medium.

Negative

No change in medium or no growth on medium.

Biochemical Test: Bacitracin Disk Susceptibility

Intended Use: To differentiate group A beta-hemolytic streptococci from other beta-hemolytic streptococci Principle: Group A streptococci growth is inhibited by bacitracin where as other beta hemolytic streptococci are unaffected by bacitracin Reagents/Media: Sheep blood agar , bacitracin disk Positive Control: S. pyogenes Negative Control: S. agalactiae Step by Step instructions: 1. 2. 3. 4.

Split the Sheep Blood Agar plate in half. Plate a lawn of organism across one half of the blood agar, using a loop and streaking one direction, and then again at a 90 degree angle. Aseptically place bacitracin disk in center of area containing inoculum Incubate for 18-24 hours at 35C in 5-10% CO2

Test Interpretation: Positive(sensitive) Negative(resistant)

Any zone of inhibition surrounding the disk Growth right up to edge of disk

Biochemical Test: Bile Solubility Test

Intended Use: Differentiate Streptococcus pneumoniae from other alpha hemolytic streptococci Principle: Bile salts will selectively lyse Streptococcus pneumoniae when added to actively growing bacterial cells. Reagents/Media: Viable culture on BA plates and 10% sodium deoxycholate. Positive Control: Streptococcus pneumoniae Negative Control: Viridans strep or Streptococcus bovis Step by Step instructions: 1.

Add one or two drops of sodium deoxycholate to several isolated colonies on a sheep blood agar plate (can use the plate that was used for optochin testing)

2.

Allow solution to dry for 5-10 minutes.

3.

Examine colonies

Test Interpretation: Positive

Colonies are lysed and disappear, leaving only a partially hemolyzed (green) area where colony had previously been.

Negative

Colonies remain intact

Biochemical Test: CAMP

Intended Use: To differentiate Streptococcus agalactiae from other Beta-hemolytic streptococci. Principle: Streptococcus agalactiae produces CAMP factor, which enhances the lysis of sheep red blood cells by staphylococcal beta-lysin. A positive reaction can be observed in 5 to 6 hours with incubation in CO2. Reagents/Media: Beta-lysin producing S. aureus, Sheep Blood Agar plate. Positive Control: S. agalactiae Negative Control: S. pyogenes Step by Step instructions: 1.

Inoculate S. aureus along a line down the center of the agar plate.

2.

Inoculate the streptococcal isolates along a thin line about 2 cm long and perpendicular to, but not touching, the S. aureus streak.

3.

Incubate plate at 35 degrees Celsius for 18 hours.

Test Interpretation: Positive

Arrowhead-shaped area of enhanced hemolysis where the two streaks approach each other.

Negative

No enhanced hemolysis.

Biochemical Test: CAT Screen

Intended Use: To identify Moraxella catarrhalis Principle: Hydrolysis of the substrate bromo-chloro-indolyl butyrate by butyrate esterate yields a blue-green color on the disk. Reagents/Media: Butyrate Disk, Water Positive Control: Moraxella catarrhalis Negative Control: Neisseria gonorrhoeae Step by Step instructions: 1.

Remove disk from vial and place on a clean glass slide.

2.

Add one drop of distilled or deionized water to moisten the disk.

3.

Obtain a heavy, visible loopful from a culture and rub it onto the disk.

4.

Incubate at room temperature for 5 minutes.

Test Interpretation: Positive

Blue to Blue-Green Color Change

Negative

No Color Change

Biochemical Test: Catalase Test

Intended Use: Used to differentiate many bacteria, most commonly between Staphylococcus and Streptococcus spp. Principle: Catalase is an enzyme that mediates the breakdown of hydrogen peroxide H2O2 into oxygen and water, neutralizing the bactericidal effects resulting in the production of visible bubbles. Reagents/Media: Catalase Reagent: 3% Hydrogen Peroxide Solution Positive Control: Staphylococcus aureus Negative Control: Streptococcus pyogenes Step by Step instructions: 1.

Transfer a small amount of bacterial colony to a surface of clean, dry glass slide using a loop or sterile wooden stick. The colony must be from a non-blood containing agar*.

2.

Place one drop of 3% H2O2 on to the slide.

3.

Observe for a reaction within 5-10 sec.

4.

Dispose of the slide in the biohazard glass disposal container.

Test Interpretation: Positive

Bubbling within 5-10 seconds

Negative

No bubbling or very few bubbles

*Note: It is recommended that colonies to be tested with the Catalase Test be taken from non-blood containing media due to the endogenous catalase activity present in animal red blood cells. Do not introduce a metallic loop into the drop of Catalase Reagent, because this often causes a false-positive reaction.

Biochemical Test: Citrate

Intended Use: To distinguish between members of the Enterobacteriaceae family Principle: The Citrate test is used to determine the ability of bacteria to utilize sodium citrate as its only carbon source. When the bacteria are capable of metabolizing citrate (permeases), citrate is cleaved by citrate lyase to oxaloacetate and acetate. The oxaloacetate is then metabolized to pyruvate and CO2. The ammonium salts are then broken down to ammonia, which increases alkalinity. The shift in pH turns the bromthymol blue indicator in the medium from green to blue above pH 7.6. Reagents/Media: Citrate Tube Media Positive Control: Klebsiella pneumoniae Negative Control: Escherichia coli Step by Step instructions: 1. Inoculate citrate agar on the slant using a needle/loop, streaking with a colony that is 18 to 24 hours old. 2. Incubate at 35oC for 18 to 24 hours. Test Interpretation: Positive Growth on slant surface and the media blue. Negative Little or no growth on slant surface and the media with no color change. Will remain green.

Biochemical Test: DNase Agar (DNA Hydrolysis) Intended Use: To identify Serratia spp. and Moraxella catharrhalis. Principle: For detection of deoxyribonuclease activity in gram negative bacteria Reagents/Media: DNase Agar plate Positive Control: Serratia marcescens

Negative Control: Escherichia coli Step by Step instructions: 1.

Allow the agar plate to warm to room temperature before use

2.

Split the plate into 2 halves.

3.

Utilizing an 18-24 hour old isolated colony of the organism to be tested, inoculate the media with a single heavy streak on one half of the plate.

4.

Incubate at 35C for 18-24 hours in an aerobic atmosphere.

Test Interpretation: Positive

Development of a rose-pink zone of color around the growth

Negative

No development of a zone of color

Biochemical Test: Hippurate hydrolysis Intended Use: Presumptive identification of group A, B, and D Streptococci Principle: Based on the ability to hydrolyze sodium hippurate. Reagents/Media: Hippurate disks, Ninhydrin reagent Positive Control: Streptococcus agalactiae Negative Control: Streptococcus pyogenes

Step by Step instructions: 1.

Dispense 0.1 mL sterile distilled or deionized water into a small tube (13 x 100 mm)

2.

Completely emulsify a large loopful of test organism in the water and shake or vortex to form a smooth suspension.

3.

Add one Hippurate disk to the suspension using aseptic technique.

4.

Incubate the tube in incubator at 35 - 37°C for 2 hrs.

5.

Following incubation, add 0.2 mL (5 drops) ninhydrin reagent to each tube and shake gently.

6.

Reincubate the tube for 10 - 15 min and read reaction.

7.

Observe and record results.

Test Interpretation: Positive

Formation of a purple-blue color in the solution

Negative

Absence of a color change in the solution.

Biochemical Test: Lysine Iron Agar slant (LIA) Intended Use: Used for differentiating certain Enterobacteriaceae, especially Salmonella spp. Principle: Lysine is present in the agar to detect the presence of lysine decarboxylase or lysine deaminase. Hydrogen sulfide production is detected by ferric ammonium citrate and sodium thiosulfate indicators. Fermentation of dextrose is detected with bromcresol purple pH indicator. Reagents/Media: Lysine Iron Agar slant Controls:

Lysine decarboxylation and H2S production: Salmonella typhimurium Lysine deamination and negative for H2S: Proteus mirabilis Dextrose fermentation (negative reaction): Shigella flexneri Step by Step instructions: 1.

Obtain Lysine Iron Agar slant

2.

Using inoculating needle, stab the butt and streak the slant

3.

Incubate with loosened cap for 18-48 hours at 35 ± 2°C

Test Interpretation: Positive: Lysine decarboxylation Alkaline (purple) butt and alkaline (purple) slant Positive: Lysine deamination

Red slant, acidic (yellow) butt

Positive: H2S production

Formation of black precipitate

Negative

Alkaline (purple) slant and acidic (yellow) butt, indicating dextrose fermentation only

Biochemical Test: Methyl Red Intended Use: To identify bacteria capable of mixed acid fermentation. Principle: The products of mixed acid fermentation are a complex mixture of acids reducing the pH of the media. Reagents/Media MRVP broth, Methyl Red Positive Control: Salmonella typhimurium Negative Control: Klebsiella pneumoniae

Step by Step instructions: 1.

Using a sterile loop, inoculate unknown organism into fresh sterile broth.

2.

Incubate the inoculated broths at 37 C for two days.

3.

After incubation, split each tube into two tubes (one for MR, one for VP)

4.

Add 5 drops of Methyl Red and observe for color change.

Test Interpretation: Positive

Red color change

Negative

No color change

Biochemical Test: MIO (Motility, Indole, Ornithine Decarboxylase) Intended Use: Determine Motility, Indole, and Ornithine Decarboxylase in Gram Negative Bacilli Principle: Organisms that have the enzyme, tryptophanase, break down tryptophan which produces indole. Indole reacts with Kovac’s Reagent (p-dimethylaminobenzaldehyde) to produce a red band on top of the media. If an organism ferments dextrose the pH of the media will go down and Bromcresol purple will react as a pH indicator. With a lower pH the indicator will change to yellow. The acid will cause enzyme activation. The enzyme will decarboxylate ornithine and diamine putrescine is produced. Diamine putrescine makes the media more alkaline and changes the media to dark purple. Reagents/Media: MIO Media, Kovac’s Reagent Positive Control: Escherichia coli

Negative Control: Klebsiella pneumoniae Step by Step instructions: 1.

Obtain a single isolated colony using a needle and stab the media through the center about half way down.

2.

Incubate aerobically at 35° C for 18-24 hours with a loose cap.

3.

Examine for motility and the production of ornithine.

4.

Add a few drops of Kovac’s Reagent and look for the production of indole.

Test Interpretation: Motility Positive

Turbidity expands beyond the inoculation line

Motility Negative

No turbidity beyond the inoculation line

Ornithine Positive

Dark purple turbidity in the media

Ornithine Negative

Yellow throughout the media

Indole Positive

When a pink to red band appears at the top of the media after Kovac’s been added

Indole Negative

When a yellow band appears on the top of the media after Kovac’s been added

Biochemical Test: Nitrate Reduction Intended Use: To determine the ability of an organism to reduce nitrate. Principle: The addition of Reagents A and B indicate the reduction of nitrate to nitrite through the production of a red color. If needed, the addition of Reagent C, zinc dust, there after determines if nitrate has been reduced further into nitrogen gas. Reagents/Media: Nitrate broth, Reagent A (sulfanilic acid), Reagent B (1,6-Cleve’s Acid), Reagent C (zinc dust) Positive Control: Escherichia coli Negative Control: Acinetobacter spp. Step by Step instructions:

1.

Inoculate nitrate broth with organism of interest.

2.

Incubate at 35°C for 24-48 hours.

3.

Add 5 drops of Reagent A and 3 drops of Reagent B. Gently swirl to mix reagents.

4.

Look for the development of a red color within 1-2 minutes.

5.

If no red color develops, add Reagent C.

Test Interpretation: Reduction of nitrate to nitrite (Positive)

Red color develops after addition of Reagents A and B.

No nitrate reduction

No red color develops after addition of Reagents A and B, but a red color develops after addition of Reagent C.

(Negative) Reduction of nitrate to nitrogen gas

No red color development after addition of Reagents A and B or Reagent C.

Biochemical Test: Ortho-Nitrophenyl-β-D-Galactopyranosidase Test (ONPG) Intended Use: To determine whether the organism is a delayed Lactose Fermenter (one that lacks the enzyme β-galactosidase permease but possesses β-galactosidase) or a true Non-Lactose Fermenter (NLF). To differentiate members of the Enterobactericeae and other microorganisms based on beta-galactosidase activity. Principle: ONPG is similar to lactose but is more readily transported through the bacterial plasma membrane and does not require β-galactosidase permease. β-galactosidase hydrolyzes ONPG, a colorless compound, into galactose and o-nitrophenol, a yellow compound. ONPG remains colorless if the organism is a NLF. Reagents/Media: Sterile Saline, commercially prepared ONPG disks or tablets Positive Control: Escherichia coli

Negative Control: Proteus mirabilis Step by Step instructions: 1.

Use a loop to transfer bacteria from pure 18-24 hour culture to a test tube containing 0.3 ml of 0.85% sterile saline. The resulting suspension should be approximately equivalent in density to a McFarland 3 opacity standard.

2.

Add a single ONPG disk to the dense bacterial suspension. Upon the addition of the disk the bacterial suspension will be clear.

3.

Incubate at 37°C and check hourly, for up to 4 hours, for the development of a yellow color change.

4.

Incubate any negative reactions (colorless) for 24 hours. Observe after 24 hours for possible delayed reactions of late lactose-fermenters.

Test Interpretation: Positive

Yellow broth color

Negative

No color change

Biochemical Test: Optochin disk susceptibility Intended Use: identification of Streptococcus pneumoniae from other alpha-hemolytic streptococci Principle: S. pneumoniae growth is inhibited by optochin whereas other alpha-hemolytic streptococci are not inhibited Reagents/Media: Sheep Blood Agar, optochin disk Positive Control: Streptococcus pneumoniae Negative Control: viridans streptococci Step by Step instructions: 1.

Split the Sheep Blood Agar plate in half

2. 3. 4. 5.

Plate a lawn of organism across half of the agar plate, using a loop and streaking one direction, and then again at a 90 degree angle. Aseptically place optochin disk in center of area containing inoculum Incubate for 18-24 hours at 35C aerobically. NOTE: This plate, after incubation and observation, can be used for the bile solubility test.

Test Interpretation: Positive(sensitive) Negative(resistant)

zone of inhibition surrounding the disk (14mm or greater) growth right up to edge of disk

Biochemical Test: Oxidase Test Intended Use: The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain. The oxidase test may be used in the presumptive identification of Neisseria spp. and in the differentiation of gram-negative bacilli. Principle: When present, the cytochrome c oxidase oxidizes the reagent (tetramethyl-p-phenylenediamine) to (indophenols), producing a purple color product. Reagents/Media: Sterile swabs, Oxidase reagent Positive Control: Pseudomonas aeruginosa Negative Control: Escherichia coli

Step by Step instructions: 1.

Remove swab from container without touching the tip.

2.

Use the swab to carefully collect 3-4 well isolated colonies from a Sheep Blood Agar plate.

3.

Drop a small drop of oxidase reagent to the swab containing colonies of interest.

4.

Analyze for color change within 30 seconds.

Test Interpretation: Positive

Blue/purple color change within 30 seconds.

Negative

No color change

*Note: The oxidase test must be performed from 5% Sheep blood agar or another medium without a fermentable sugar, otherwise a false negative result may occur.

Biochemical Test: Phenylalanine Deaminase Intended Use:...