Microbiology Lab Identification of Unknown Tests21 cards

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1

Oxidase test

  • Tests for the presence of cytochrome c or cytochrome a3- electron transport chain
  • REAGENTS:
  •         1% tetramethyl-p-phenylenediamene dihydrochloride
  •      positive control– Pseudomonas aeruginosa
  •      negative control– Escherichia coli
  • RESULTS:
  •         dark blue/purple (positive) within 15 sec.                           No color change (negative) within 15 sec.

2

Sugar fermentation test

  • Tests for the fermentation of glucose, lactose, sucrose and mannitol. Also tests for gas production
  • REAGENTS:
  •       -pH indicator: Phenol Red for detecting acid production upon fermentation; pH of media will drop as acid is produced; (red for pH >7 and yellow for pH <7)
  •       -inverted tube (Durham tube): for detecting gas production upon fermentation; if a bubble appears in the tube, then gas was produced
  • RESULTS:
  •       - positive: yellow color change for sugar fermentation; bubble in the tube for gas production
  •       - negative: red color for no sugar fermentation; no bubble in the tube for no gas production

3

Nitratase Test

Tests for: the presence of nitrate as an electron acceptor in anaerobic respiration and the presence of nitratase which converts nitrate to nitrite
REAGENTS:
        - A: sulfanilic acid
        - B: dimethyl-α-naphthylamine
Add 10-15 drops of each reagent to the tube of Nitrate broth, vortex, and look for color change. If no color change occurs, then add zinc, and look for color change.
RESULTS:
        - positive: red color change initially or no color change once zinc is added (due to nitrite being broken down further into other products)
        -negative: no color change initially; confirmed by adding zinc which acts like nitratase and causes the red color change

4

Catalase Test

Tests for: presence of the enzyme catalase which breaks down toxic oxygen compounds like hydrogen peroxide (H2O2) into water and oxygen
REAGENTS:
        -hydrogen peroxide (3%H2O2)
     Students will be performing the Catalase Test using the TSA slant of their unknown. Add several drops of 3% H2O2 to the TSA slant, allowing the reagent to flow down the slant over the bacteria, and watch for the production of bubbles.
RESULTS:
         -positive: bubbles---> Catalase present
         -negative: no bubbles

5

Methyl Red Test (MR)

Tests for: the production of acids upon glucose fermentation; an indicator that the organism is a mixed acid fermenter
REAGENTS:
      - Methyl Red: a pH indicator (red for pH<5.0 and yellow for pH> 5.0); for     detecting acidic products of glucose fermentation
   Add 10-15 drops of Methyl Red to one of the two tubes of MRVP media inoculated with the unknown bacterium, vortex and look for a color change.
RESULTS:
        -Positive: red color change
        -Negative: yellow color

6

Voges- Proskauer Test

Tests for: the production of alcohols (2,3-butanediol and ethanol) upon glucose fermentation; detects the pre-cursor of 2,3-butanediol, acetoin
REAGENTS:
        -Barritt’s Reagents:
              A: alpha-naphthol
              B: KOH (potassium hydroxide)
Add 10-15 drops of alpha-naphthol to the other of the two tubes of MRVP media inoculated with the unknown bacterium, vortex, add 10-15 drops of KOH, vortex intermittently for 10-15 minutes, and look for a color change.
RESULTS:
         -positive: red color change
         -negative: yellow color

7

Simmons Citrate Test

Tests for: utilization of citrate as the sole carbon source
RESULTS:
      -positive: Prussian Blue color change--->Citrate used as sole source
      -negative: green color

8

Phenylalanine Deamination

Tests for: the presence of phenylalanase which breaks down phenylalanine into phenylpyruvic acid (PPA)
REAGENTS:
        -10% ferric chloride; detects the PPA
RESULTS:
        -positive: deep green color change
        -negative: yellow (color of the reagent)

9

SIM Media Deep

Tests for: the presence of H2S production (produced as a product of cysteine deamination), the presence of tryptophanase which can hydrolyze tryptophan into indole and pyruvic acid, and motility.
REAGENTS:
         -motility: semi-solid media
         -iron salts in media: used to test for H2S production
   chloroform and Kovac’s Reagent (p-dimethyl aminobenzaldehyde): used to test for the presence of tryptophanase by detecting indole
RESULTS:
     -positive:
           -H2S production: dark precipitate of iron sulfide forms
            -presence of tryptophanase: red ring at the top of the media
            -motility: growth radiating from the line of inoculation; cloudy throughout the tube
     -negative:
           -H2S production: no dark precipitate of iron sulfide forms
           -presence of tryptophanase: no red ring at the top of the media
         -motility: growth only along the line of inoculation

10

Kigler's Iron Agar Slant Test

Tests for: the presence of H2S production (produced as a product of cysteine deamination), gas production, and fermentation of glucose and lactose
REAGENTS:
        - Iron salts: used to test for H2S production
         - Phenol Red: used as a pH indicator to detect acid production upon glucose and/or lactose fermentation; (red for pH>7, and yellow for pH <7);
due to the different percentages of glucose (0.1% w/v) and lactose (1% w/v) in the media, you get a different distribution of color change in the media
RESULTS:
    - positive:
           -gas production: cracks in the media; or a bubble of air at the bottom of the tube which may push the media up in the tube
           -H2S production: dark precipitate of iron sulfide forms (black color in media)
           -Glucose fermentation: slant remains red; yellow in bottom of tube
           -Lactose fermentation: yellow throughout the tube
-negative:
           -gas production: no cracks in the media
         -H2S production: no dark precipitate of iron sulfide forms
         -Glucose fermentation: red in bottom of tube
         -Lactose fermentation: red throughout the tube

11

Urea Hydrolysis Test

Tests for: the presence of urease which can hydrolyze urea into ammonia and CO2
REAGENTS:
       -pH indicator: Phenol Red for detecting ammonia production; pH of media will rise as ammonia is produced; (red for pH >7 and yellow for pH <7)
RESULTS:
     - positive: red (fuschia) color change
     - negative: yellow (orange) color

12

Tryptone Hydrolysis

Tests for: presence of tryptophanase which can hydrolyze tryptophan into indole and pyruvic acid
REAGENTS:
       -Kovac’s Reagent (p-dimethylaminobenzaldehyde); used for detecting indole
RESULTS:
       - positive: red ring at the top of the media
       - negative: no red ring

13

Fat Hydrolysis (Spirit Blue Agar)

Tests for: the presence of lipases which can hydrolyze fats into glycerol and fatty acids
REAGENTS:
      -media: Spirit Blue Agar (which contains lipids)
RESULTS:
      -positive: depletion of the lipids around the line of inoculation (zone of hydrolysis) or presence of a dark precipitate on the growth which results from the lowering of the pH
      -negative: no dark precipitate on growth; no depletion of lipids

14

Casein Hydrolysis (Milk Agar)

Tests for: the presence of caseinase which can hydrolyze casein (the predominant protein in milk that gives it its white color)
REAGENTS:
        - media: Milk Agar
RESULTS:      
       -positive: clear zone around growth (zone of hydrolysis)
       -negative: no zone round growth (remains milky-white)

15

Starch Hydrolysis

Tests for: the presence of amylases which can hydrolyze starch
REAGENTS:
       -media: starch agar
       - iodine
RESULTS:
       - positive: clear zone around growth (zone of hydrolysis)
       - negative: no clear area around growth and media is stained brownish-blue by the starch-iodine complex

16

Klebsiella Pneumonia

STAIN:
     -Capsular: Figure "8" Capsule
SPECIAL MEDIA:
     -MCA: Red
     -EMB: Pink
DISEASE: NONE!



17

Staphylococcus Epidermidis

STAIN:
     -Capsular: no capsule
     -Acid Fast: Negative
     -Gram: Positive
     -Spore: no endospore
SPECIAL MEDIA:
     -PEA: growth
     -MSA: Pink
     -MCA: no growth
COMMENTS:
     -Non-motile

18

Staphylococcus Aureus

STAIN: none
SPECIAL MEDIA:
     -MSA: yellow
     -EMB: No growth

19

Mycobacterium Tuberculosis

STAIN:
     -Acid-Fast: Positive
     -Gram: Positive
DISEASE:
     -Tuberculosis
COMMENTS:
     -Slow growth (8 weeks)

20

Mycobacterium Leprae

STAIN:
     -Acid-Fast: Positive
     -Gram: Positive
DISEASE:
     -Leprosy
COMMENTS:
     -Grows on armadillo feet only (no artificial media)

21

Mycobacterium Smegmatis

STAIN:
     -Acid-Fast: Positive
     -Gram: Positive