Microbio LAB Final Exam

Starch Hydrolysis

Amylose ———-(Amylase)—————> Mono- and Di- saccharides

Test

• Starch Hydrolysis (Amylase Test)

Media

• Starch agar plate
  (Contains soluble starch)

Substrate

• Starch (a polysaccharide)

Enzyme

• Amylase
  (Also sometimes amylopectinase)

End Products

• Smaller carbohydrates:

  • Maltose

  • Glucose

  • Dextrins

Other Reagents

• Iodine (flood the plate after incubation)
  → Iodine binds to intact starch, producing a blue‑black color

Interpretations

• Positive:
  Clear zone around bacterial growth after adding iodine
  → organism produced amylase and hydrolyzed starch

• Negative:
  Blue‑black color right up to the edge of growth
  → starch remains intact; no amylase produced

Troubleshooting

• Don’t add iodine before incubation: it will kill the bacteria

• Thin streaking helps: thick growth can hide clearing zones

• Old plates dry out: can cause false clearing

• Under‑incubation: weak amylase producers may appear negative

• Uneven iodine flooding: can create patchy color and misinterpretation

DNAase

DNA ————-(DNAase)———→ Nucleotides

Test

• DNase Test

Media

• DNase agar containing:

  • DNA

Substrate

• DNA (deoxyribonucleic acid)

Enzyme

• DNase (deoxyribonuclease)

End Products

• Small DNA fragments

Other Reagents

• HCl

Interpretations

• Positive:
  Clear, colorless zone around growth where DNA has been hydrolyzed and HCl is released
  → organism produces DNase

• Negative:
  No clearing
  → organism does not produce DNase

Troubleshooting

• Weak or no growth: organism may appear negative; ensure proper inoculation and incubation

• Over‑inoculation: thick streaks can mask clearing; use a thin, straight line streak

Carbohydrate Fermentation

Sugar ——————> Acid end products ± gas

Test

• Detects acid ± gas from fermentation of a specific carbohydrate.

Media

• Phenol red carbohydrate broth

  • Peptone, beef/yeast extract

  • Single carbohydrate (glucose, lactose, sucrose, etc.)

  • Phenol red (pH indicator)

  • Durham tube (gas detection)

Substrate

• The specific carbohydrate added to the broth.

Enzyme

• Carbohydrate‑specific permeases

• Fermentative enzymes (glycolysis → fermentation pathways)

End Products

• Acid → yellow

• Acid + gas → yellow + bubble

• Alkaline (peptone use) → pink/fuchsia

Other Reagents

• None added after incubation; indicator is built‑in.

Interpretations

• Yellow, no bubble → Acid only (A)

• Yellow + bubble → Acid + gas (A/G)

• Red/orange → No fermentation (–)

• Pink/fuchsia → Alkaline (K)

Troubleshooting

• Reversion (yellow → red/pink): read at 18–24 hrs.

• Weak color change: low inoculum or slow fermenter.

• No gas: Durham tube not fully filled or gas dissolved.

• All tubes yellow: contamination or incorrect pH.

TSI

H2S + Fe ++ ———————> FeS

Test

• Triple Sugar Iron (TSI) Agar Test

Media

• TSI agar containing:

  • Three sugars: glucose (0.1%), lactose (1%), sucrose (1%)

  • Peptones

  • Phenol red (pH indicator)

  • Ferrous sulfate (H₂S indicator)

  • Sodium thiosulfate (sulfur source)

Substrate

• Glucose

• Lactose

• Sucrose

• Sodium thiosulfate (for H₂S production)

• Peptones (used when sugars are depleted)

Enzyme

Not a single enzyme — the test detects multiple metabolic pathways, including:

• Carbohydrate‑fermenting enzymes

• Thiosulfate reductase (for H₂S production)

• Deaminases (for peptone utilization under aerobic conditions)

End Products

• Depending on organism:

  • Acids (yellow color)

  • Alkaline products (red color)

  • Gas (bubbles, cracks, lifting of agar)

  • H₂S (black precipitate of ferrous sulfide)

Other Reagents

• none

Interpretations

Color Key:

• A = Acid (yellow)

• K = Alkaline (red)

• H₂S = black precipitate

• G = gas

Common Patterns:

• K/A: Glucose fermentation only

  • Slant red, butt yellow

• A/A: Glucose + lactose and/or sucrose fermentation

  • Entire tube yellow

• K/K: No sugar fermentation; peptone utilization

  • Entire tube red

• Black precipitate: H₂S production

  • Butt is automatically acidic (A) even if obscured

• Gas: Cracks, bubbles, or agar lifted

Troubleshooting

• Read at 18–24 hours:

  • Reading too late → glucose‑only fermenters may revert slant to K/A → K/K (false negative)

• Black butt hides color:

  • Assume A (acid) in the butt if H₂S is present

• Shallow stabbing:

  • May falsely appear K/K because butt didn’t get inoculated

• Over‑incubation:

  • Peptone reversion on slant can mask true fermentation

• Dry agar:

  • can mimic cracks or gas production

Urea Hydrolysis

Urea———(Urease)———→ Ammonia and CO2

Test

• Urea Hydrolysis (Urease Test)

Media

• Urea broth or urea agar containing:

  • Urea

  • Phenol red (pH indicator)

  • Peptones

  • Potassium phosphate buffer

Substrate

• Urea

Enzyme

• Urease

End Products

• Ammonia (NH₃)

• Carbon dioxide (CO₂)
  Ammonia raises pH → alkaline environment

Other Reagents

• None

Interpretations

• Positive:
  Hot pink / fuchsia color
  → rapid urease activity → ammonia produced → pH ↑
  Common in Proteus, Morganella, Klebsiella

• Negative:
  Yellow or peach color
  → no urease or very slow urease activity

• Weak/slow positive:
  Slight pink after 24–48 hours
  → organism produces urease slowly

Troubleshooting

• Read at correct time:

  • Rapid urease producers (Proteus) turn pink within 2–6 hours

  • Reading too early may miss slow positives

• Over‑incubation:

  • Some organisms alkalinize media from peptone use → false pink

• Broth vs. agar:

  • Broth is more sensitive; agar may miss weak urease producers

• Heavy inoculum:

  • Can cause uneven color or false positives

• Old media:

  • Urea breaks down over time → unreliable results

MacConkey Agar

Lactose ————————> Acid end products

Test

• MacConkey Agar (MAC) — Selective & Differential Medium

Media

• Contains:

  • Bile salts (selective)

  • Crystal violet (selective)

  • Lactose (differential substrate)

  • Neutral red (pH indicator)

  • Peptones

Substrate

• Lactose

Enzyme

• β‑galactosidase
  (Enzyme used by lactose‑fermenting bacteria)

End Products

• Acidic fermentation products → lower pH → colony color change

Other Reagents

• None

Interpretations

• Growth:
  Organism is Gram‑negative (or Gram‑variable)
  Bile salts + crystal violet inhibit Gram‑positives

• Pink/red colonies:
  Lactose‑positive (fermenter)
  → acid produced → neutral red turns colonies pink
  Examples: E. coli, Klebsiella, Enterobacter

• Colorless/tan colonies:
  Lactose‑negative (non‑fermenter)
  Examples: Proteus, Salmonella, Shigella, Pseudomonas

• Precipitated bile around colonies:
  Strong lactose fermenter (e.g., E. coli)

Troubleshooting

• Weak growth:
  Could be due to Gram‑positive organism or stressed cells

• Over‑incubation:
  Acid may diffuse → plate background becomes pinkish

• Dry plates:
  Can distort colony color

• Mixed cultures:
  Pink and colorless colonies may appear together → isolate single colonies

• Misreading color:
  Look at colony color, not the agar itself

Indole

tryptophan + H2O ——--(tryptophanase)—→ indole + pyruvate + ammonia

Test

• Indole Test (Tryptophan Hydrolysis)

Media

• Tryptone broth (rich in tryptophan)

• Can also be part of SIM medium (Sulfide–Indole–Motility)

Substrate

• Tryptophan

Enzyme

• Tryptophanase

End Products

• Indole

• Pyruvate

• Ammonia (NH₃)

Other Reagents

• Kovac’s reagent (p‑dimethylaminobenzaldehyde + HCl + amyl alcohol)
  Added after incubation

Interpretations

• Positive:
  Cherry‑red / bright red ring at the top after adding Kovac’s
  → indole produced → organism has tryptophanase
  Common positives: E. coli, Proteus vulgaris

• Negative:
  Yellow or no color change after adding Kovac’s
  → no indole produced

Troubleshooting

• Do NOT shake after adding Kovac’s:
  Red color forms in the alcohol layer only; shaking disperses it.

• Use tryptone broth, not nutrient broth:
  Nutrient broth has too little tryptophan → false negatives.

• Over‑incubation:
  Some organisms degrade indole over time → weak or false negatives.

• SIM medium caution:

  • Black precipitate (H₂S) can obscure results.

  • Always read the top layer only.

• Too heavy inoculum:
  Can cause uneven color or delayed reaction

Methyl Red

Glucose ————→ mixed acids (pH < 4.5)

Test

• Methyl Red (MR) Test (Part of the IMViC series)

Media

• MR‑VP Broth

  • Contains glucose, peptone, and phosphate buffer

Substrate

• Glucose

Enzyme

• Mixed‑acid fermentation pathway enzymes
  (not a single enzyme—organisms use a series of enzymes to produce stable acids)

End Products

• Stable acidic end products: lactic acid, acetic acid, formic acid

• CO₂ and H₂ may also be produced

Other Reagents

• Methyl red indicator
  Added after incubation

Interpretations

• Positive (MR⁺):
  Red color after adding methyl red
  → organism performs mixed‑acid fermentation
  → produces stable acids that keep pH < 4.4
  Common positives: E. coli, Proteus, Salmonella

• Negative (MR⁻):
  Yellow or orange
  → organism does not produce stable acids
  → pH > 6.0
  Common negatives: Enterobacter, Klebsiella

Troubleshooting

• Read immediately after adding reagent:
  Color can fade or shift if left too long.

• Orange = inconclusive:
  Often means borderline pH; repeat test or extend incubation.

• Under‑incubation:
  Mixed‑acid fermenters may appear negative if acids haven’t accumulated yet.

• Do NOT shake vigorously after adding methyl red:
  Can cause uneven color distribution.

• Use MR‑VP broth only:
  Other broths lack the correct buffering system → false results

Voges-Proskaur

Glucose ————> AMC ———> Butylene glycol (4.5 > pH > 7)

Test

• Voges–Proskauer (VP) Test (Part of the IMViC series)

Media

• MR‑VP Broth

  • Contains glucose, peptone, and phosphate buffer

Substrate

• Glucose
  (specifically the acetoin precursor: α‑acetolactate)

Enzyme

• Acetoin‑producing pathway enzymes
  (2,3‑butanediol fermentation pathway)

End Products

• Acetoin (detected intermediate)

• 2,3‑butanediol (final product)

Other Reagents

Added after incubation:

• VP Reagent A: α‑naphthol

• VP Reagent B: 40% KOH
  (Often added in a 1:3 ratio, A then B)

Interpretations

• Positive (VP⁺):
  Red color within 10–30 minutes
  → organism produces acetoin via 2,3‑butanediol fermentation
  Common positives: Enterobacter, Klebsiella, Serratia

• Negative (VP⁻):
  No color change or copper/brown
  → no acetoin production
  Common negatives: E. coli, Proteus, Salmonella

Troubleshooting

• Timing matters:
  Red color may take up to 30 minutes; reading too early gives false negatives.

• Copper color ≠ positive:
  Copper is a reaction between reagents, not acetoin.

• Shake after adding reagents:
  Oxygen is required for the color reaction; gentle mixing improves accuracy.

• Use MR‑VP broth only:
  Incorrect media → weak or false results.

• Over‑incubation:
  Can reduce acetoin levels → weak positives.

Citrate

Citrate————→ alkaline end products

Test

• Citrate Utilization Test
  (Part of the IMViC series)

Media

• Simmons Citrate Agar

  • Contains sodium citrate (sole carbon source)

  • Ammonium dihydrogen phosphate (sole nitrogen source)

  • Bromothymol blue (pH indicator)

• Substrate

• Citrate (carbon source)

• Ammonium phosphate (nitrogen source)

Enzyme

• Citrate permease
  (transport enzyme that allows citrate uptake)

End Products

• Alkaline products: ammonia (NH₃) + ammonium hydroxide (NH₄OH)

• These raise the pH → indicator turns blue

Other Reagents

• None

Interpretations

• Positive (Citrate⁺):
  Blue slant (Prussian blue)
  OR visible growth even if medium stays green
  → organism can use citrate as sole carbon source
  Common positives: Enterobacter, Klebsiella, Salmonella

• Negative (Citrate⁻):
  Green with no growth
  → organism cannot use citrate
  Common negatives: E. coli

Troubleshooting

• Growth alone = positive:
  Some organisms grow before the color changes; don’t rely only on blue.

• Avoid heavy inoculum:
  Too much bacteria can carry over carbon → false positives.

• Use a light streak on the slant:
  Stabbing the butt is incorrect and may distort results.

• Incubation time matters:
  Weak citrate users may need longer to show color change.

• Check for drying: Over‑dry agar can cause false blue at the edges

Mannitol Salt Agar

Mannitol ————→ Acid end products

Test

• Mannitol Salt Agar (MSA) Test
  Selective + Differential medium

Media

• Mannitol Salt Agar containing:

  • 7.5% NaCl (high salt)

  • Mannitol (carbohydrate)

  • Phenol red (pH indicator)

  • Peptones

Substrate

• Mannitol (for fermentation)

• Peptones (if mannitol not used)

Enzyme

• Mannitol‑fermentation pathway enzymes
  (not a single enzyme—organisms use a set of enzymes to ferment mannitol)

End Products

• If mannitol is fermented:

  • Acidic end products → lower pH → yellow color

• If mannitol is NOT fermented:

  • Alkaline products from peptone use → pink/red color

Other Reagents

• None
  (no reagents added after incubation)

Interpretations

• Growth = salt tolerant
  (Organism can survive 7.5% NaCl)

• Yellow colonies + yellow medium:
  Mannitol fermentation positive
  → acid produced
  → Staphylococcus aureus is the classic positive

• Pink/red colonies, medium stays red:
  Mannitol fermentation negative
  → organism grows but does not ferment mannitol
  → Staphylococcus epidermidis typical

• No growth:
  Not salt tolerant
  → most Gram‑negative bacteria and many Gram‑positives

Troubleshooting

• Weak yellowing:
  Could be early fermentation—extend incubation for clarity.

• Over‑incubation:
  Acid can diffuse unevenly; read plates at recommended time.

• Heavy inoculum:
  Can cause false yellowing from metabolic byproducts; streak lightly.

• Dry plates:
  Dehydration can cause edge color artifacts—ignore edges.

• Color confusion:
  Phenol red:

  • Yellow = acidic

  • Red = neutral

  • Hot pink = alkaline

Catalase

2 H2O2 —————-(Catalase)———> 2H2O + O2

Test

• Catalase Test
  (Detects the presence of the enzyme catalase)

Media

• No special medium required

  • Grown on any nutrient-rich agar (NOT blood agar for the test itself)

Substrate

• Hydrogen peroxide (H₂O₂)

Enzyme

• Catalase

End Products

• Water (H₂O)

• Oxygen gas (O₂) → visible bubbles

Other Reagents

• 3% Hydrogen peroxide (H₂O₂)
  Added directly to bacterial colony

Interpretations

• Positive:
  Immediate bubbling
  → organism produces catalase
  → breaks down H₂O₂ into O₂
  Common positives: Staphylococcus spp., Micrococcus spp.

• Negative:
  No bubbles
  → organism lacks catalase
  Common negatives: Streptococcus spp., Enterococcus spp.

Troubleshooting

• Do NOT use colonies from blood agar:
  RBCs contain catalase → false positives.

• Use fresh H₂O₂:
  Old peroxide loses activity → false negatives.

• Avoid metal loops:
  Metal can catalyze bubbling → false positives. Use wooden stick or plastic loop.

• Read immediately:
  Delayed bubbling is not a true positive.

• Too much culture:
  Thick clumps can trap bubbles → weak or unclear reaction

Hemolysis

Test

• Hemolysis on Blood Agar
  (Differential test for hemolytic patterns)

Media

• Blood Agar Plate (BAP)

  • Tryptic soy agar or nutrient agar base

  • 5% sheep blood

Substrate

• Red blood cells (RBCs) — specifically hemoglobin

Enzyme

• Hemolysins
  (type depends on organism; e.g., streptolysins)

End Products

• Alpha hemolysis: partial breakdown → methemoglobin (green)

• Beta hemolysis: complete breakdown → clearing

• Gamma hemolysis: no breakdown → no change

Other Reagents

• None
  (no reagents added after incubation)

Interpretations

• Alpha (α) hemolysis:
  Greenish discoloration around colonies
  → partial RBC lysis
  → Streptococcus pneumoniae, Streptococcus viridans

• Beta (β) hemolysis:
  Clear, transparent zone around colonies
  → complete RBC lysis
  → Streptococcus pyogenes, Staphylococcus aureus

• Gamma (γ) hemolysis:
  No change in medium
  → no hemolysis
  → Enterococcus faecalis, some Staphylococcus spp.

Troubleshooting

• Incubate plates upside down:
  Prevents moisture from pooling and distorting hemolysis zones.

• Don’t misread alpha as gamma:
  Alpha is subtle—look for green, not clearing.

• Use fresh blood agar:
  Old plates can dry out → false patterns.

• Stab technique for streptolysin O:
  Oxygen‑labile hemolysins may show stronger beta hemolysis under the agar.

• Avoid thick streaks:
  Heavy inoculum can mask hemolysis zones.

• Incubation atmosphere matters:
  CO₂ can enhance alpha hemolysis in some species.

The difference between cell and colony morphology.

Cell Morphology (Microscopic)

What it describes:
The shape, size, and arrangement of individual bacterial cells when viewed under a microscope.

How it’s observed:

• Gram stain

• Simple stain

• Wet mount

• Oil immersion (1000×)

What you report:

• Shape: cocci, bacilli, spirilla, vibrio

• Arrangement: chains, clusters, pairs, tetrads

• Gram reaction: + or –

• Cell size

• Special structures: endospores, capsules, flagella

Think: What a single bacterium looks like up close.

Colony Morphology (Macroscopic)

What it describes:
The appearance of a bacterial colony growing on solid media (agar plate).

How it’s observed:

• Naked eye

• Dissecting microscope (optional)

What you report:

• Form: circular, irregular, filamentous

• Elevation: flat, raised, convex, umbonate

• Margin: entire, undulate, lobate, curled

• Color: pigment production

• Texture: smooth, rough, mucoid, dry

• Opacity: transparent, translucent, opaque

• Size: pinpoint, small, large

• Hemolysis (on blood agar)

Think: What a whole population looks like when it grows together.

Cell morphology = microscopic shape/arrangement of individual cells.
Colony morphology = macroscopic appearance of bacterial growth on agar.

Names and purposes of commonly used lab equipment

1. Microscope

Purpose: Magnifies small specimens (bacteria, cells) for detailed observation.

2. Microscope Slides & Cover Slips

Purpose: Hold specimens for staining and microscopic examination.

3. Inoculating Loop / Inoculating Needle

Purpose: Transfers bacteria; used for streaking plates, inoculating broths, and picking colonies.

4. Bunsen Burner

Purpose: Provides flame for sterilizing loops, needles, and creating aseptic zones.

5. Incubator

Purpose: Maintains optimal temperature for microbial growth (usually 35–37°C).

6. Autoclave

Purpose: Sterilizes media, tools, and waste using high‑pressure steam.

7. Petri Dishes (Agar Plates)

Purpose: Grow and isolate bacterial colonies on solid media.

8. Test Tubes (Broth or Slant Tubes)

Purpose: Grow bacteria in liquid or solid slant media.

9. Pipettes (Micropipettes / Serological Pipettes)

Purpose: Accurately measure and transfer liquids.

10. Pipette Tips

Purpose: Disposable tips used with micropipettes to prevent contamination.

11. Vortex Mixer

Purpose: Mixes samples quickly and evenly.

12. Centrifuge

Purpose: Separates components by spinning (pelleting cells, separating supernatant).

13. Hot Plate / Stir Plate

Purpose: Heats or stirs solutions.

14. Water Bath

Purpose: Maintains stable temperatures for incubation or melting agar.

15. pH Meter / pH Strips

Purpose: Measures acidity or alkalinity of solutions.

16. Forceps / Tweezers

Purpose: Handle small objects or sterile materials.

17. Beakers & Flasks (Erlenmeyer, Florence)

Purpose: Hold, mix, or heat liquids.

18. Graduated Cylinder

Purpose: Measures liquid volumes accurately.

19. Disposable Gloves

Purpose: Protects hands and prevents contamination.

20. Lab Coat

Purpose: Protects clothing and skin from spills.

21. Safety Goggles

Purpose: Protects eyes from splashes and chemicals.

22. Biohazard Bags / Sharps Container

Purpose: Safe disposal of contaminated materials and sharp objects.

23. Staining Rack

Purpose: Holds slides during staining procedures.

24. Wash Bottle (usually distilled water)

Purpose: Rinses slides, adds water, or cleans glassware.

25. Spectrophotometer

Purpose: Measures bacterial density (optical density at 600 nm

Media types and the selective and/or differential agents

Medium	Type	Selective Agents	Differential Agents
Blood Agar	Enriched + Differential	None	RBCs (hemolysis)
Chocolate Agar	Enriched	None	None
MacConkey	Selective + Differential	Bile salts, crystal violet	Lactose, neutral red
MSA	Selective + Differential	7.5% NaCl	Mannitol, phenol red
DNase	Differential	None	DNA, methyl green
TSI	Differential	None	Sugars, phenol red, FeSO₄
Carb Fermentation	Differential	None	Carb + phenol red + Durham tube
EMB	Selective + Differential	Eosin Y, methylene blue	Lactose, dyes
HE	Selective + Differential	Bile salts	Sugars, indicators, Fe salts

Immunotechnology theory and terms

Nitrate Test

Reduction is defined as the removal of electrons. Sometimes, this involves the removal of oxygen or hydrogen from a compound. A nutrient broth with nitrate (NO 3 ) added is termed a nitrate broth. There are three possible ways bacteria may react to the presence of nitrate in a medium:

• Some bacteria cannot use nitrate at all. Therefore, the nitrate remains unchanged in the medium. These organisms lack the enzymes for nitrate reduction.

• Some bacteria convert the nitrate (NO 3 ) to nitrite (NO 2). These bacteria only have a single enzyme, and the biochemical reaction is shown here:

Nitrate ———(nitrate reductase)———> Nitrite

Some bacteria have an additional enzyme that further reduces nitrite to nitrogen gas (N2 ) or ammonia (NH 3 ).

Nitrate ———(nitrate reductase)———> Nitrite——-(nitrite reductase)———>N2 or NH3

Nitrate Reduction Method

1) Inoculate a tube of nitrate broth, or Indole-nitrate broth with the test bacteria, using standard aseptic techniques.

2) Incubate 24-48 hours.

3) Following incubation, add 16 drops of sulfanilic acid and 16 drops of alpha- napthylamine (Note: this is not the same as alpha-napthol) to the tube. These reagents test for the presence of nitrate.

a. If the addition of these reagents cause the media to turn red, the organism reduced nitrate to nitrite, and has only one enzyme. You have completed the test, and it is positive for nitrate reductase.

b. If nothing happens upon the addition of these reagents, there are two possibilities. First, the bacteria may have done nothing to the nitrate. Second, the bacteria may have reduced all the available nitrate to nitrite, and then reduced all of the nitrite to either nitrogen gas or ammonia. To determine which is it, move to step 4.

4) Add a small quantity of zinc dust to the same tube. Zinc serves as a catalyst to reduce nitrate to nitrite. Remember, you have already added reagents to the tube that will show a color change to red if there is nitrite present. So, if you add zinc to the media, and it turns red, that means there was nitrate in the media that just got turned to nitrite due to the addition of zinc. This means the bacteria never used the nitrate, and is negative for nitrate reductase.

5) If the media did not turn red when you added zinc, that means the bacteria reduced all available nitrate to nitrite, and then also reduced all available nitrite to either ammonia or nitrogen gas. This is considered a positive reaction for full nitrate reduction.

Motility Testing: Hanging Drop Slide

Motility is defined as movement in a purposeful direction. It is best determined in a broth culture that is 24 hours old, or less.

Methods:

1) Obtain a clean depression slide, 2 toothpicks, and 2 cover slips.

2) Use the toothpicks to make a thin line of Vaseline around the depressed areas on both slides. The circle must be complete to ensure a seal, but avoid making the circle too thick, as it will obscure your vision.

3) Using proper aseptic technique, and flaming your loop each time you enter the stock culture, put 3 loops of the test broth on the center of a coverslip to form a puddle. DO NOT SMEAR.

4) Repeat step 3 on your other cover slip using a non-motile control organism provided by your instructor.

5) For each slide/cover slip, invert the slide with Vaseline on it and touch it to the coverslip so that the drops are in the center of the depression on the slide. You should now have one cover slip/slide with your unknown organism and one cover slip/slide with a non-motile control organism provided by your instructor.

6) Invert the slides again so that the coverslips are on top, and view under high power in your microscope. Keep the light low – the bacteria may prove difficult to find if the light is too bright. Using the lower power lens, locate the edge of the drop and fine focus on the edge. Then, parfocal to high power. Do not use oil immersion to view motility.

7) Compare the movement (or lack thereof) of the nonmotile control organism with your organism and determine if your organism displays motility

Oxidase Test

Oxidase Test

The purpose of this test is to detect the presence of cytochrome oxidase, which is the final component of the electron transport system, and is the enzyme that catalyzes the transfer of electrons to molecular oxygen, leading to the formation of water. Kovac’s oxidase reagent is colorless in its reduced form. However, when it is oxidized, it immediately changes to a dark blue/purple color. The presence of cytochrome oxidase indirectly causes the removal of electrons from Kovac’s reagent, causing an immediate purple reaction.

Methods

1) Grow the organism to be tested for 24-48 hours on a solid medium.

2) Obtain a small piece of paper towel, and a triangle of Whatman number 1 filter paper.

3) Place 2-3 drops of Kovac’s oxidase reagent on the filter paper.

4) To avoid contamination, place the filter paper on top of the paper towel.

5) Use aseptic technique to obtain a heavy inoculum of the test bacteria, and smear it on the reagent saturated area of the filter paper.

6) Rub hard, smearing the inoculum around on the filter paper. Cytochrome oxidase is an endoenzyme, which means you must rupture at least a few of the cells in order for it to come into contact with the reagent.

7) A dark blue or purple color will appear within 30 seconds if cytochrome oxidase is present. This is a positive reaction.

8) If the color does not develop, or begins to develop after a delay, this is a negative reaction.

9) Dispose of the paper towel and filter paper in the burn tray.