Aseptic Technique and Bacterial Culture Practice Flashcards

A set of vocabulary flashcards covering aseptic techniques, bacterial culture methods, microscopy, Gram staining, and metabolic biochemical tests.

Aseptic technique

Procedure that prevents contamination of the bacterial culture, laboratory environment, equipment, and personnel to ensure accurate experimental results.

Flaming (Inoculating Loop)

The process of sterilizing the loop before and after use to prevent contamination of the culture and the laboratory environment.

Inverted Incubation

The practice of incubating Petri dishes upside down to prevent condensation from dripping onto the agar surface.

Quadrant streak

A technique used to separate bacterial cells by dilution to allow individual cells to develop into isolated colonies.

Colony-forming unit (CFU)

One viable bacterial cell or group of cells capable of producing one visible colony on solid media.

Pure culture

A laboratory culture that contains only one species of microorganism.

Mixed culture

A laboratory culture that contains two or more species of microorganisms.

Colony morphology

The observable appearance of bacterial colonies, including size, color, shape, margin, elevation, texture, and opacity.

Wet mount

A slide preparation used to observe living microorganisms in their natural state, primarily for motility and cell arrangement.

Phase contrast microscopy

A microscopy technique that increases contrast in transparent living cells without staining to enhance visibility.

Total magnification

The product of the objective lens and ocular lens powers, calculated as $40 \times 10 = 400\times$ for a $40\times$ objective and $10\times$ ocular.

Field of view

The visible area seen through the microscope, which decreases as magnification increases.

Working distance

The physical distance between the objective lens and the slide, which decreases as magnification increases.

Immersion oil

A substance used with the $100\times$ objective to minimize light refraction and improve resolution and clarity.

Heat fixation

A step in smear preparation that kills bacteria, attaches them to the slide, and preserves morphology for staining.

Staphylococcus aureus

A Gram-positive coccus that typically appears in grape-like clusters due to division in multiple planes.

Escherichia coli

A Gram-negative bacillus (rod) that typically appears as single rods.

Crystal violet

The primary stain in the Gram stain procedure that stains all cells purple.

Gram's iodine

The mordant used in Gram staining that forms a complex with crystal violet.

Alcohol/acetone

The decolorizer in the Gram stain that removes the crystal violet-iodine complex from Gram-negative cells.

Safranin

The counterstain in Gram staining that colors decolorized Gram-negative cells pink.

Peptidoglycan

The thick cell wall component in Gram-positive bacteria that retains the crystal violet–iodine complex during decolorization.

Endospore stain

A staining procedure that typically uses older cultures because nutrient depletion and stress trigger the formation of endospores.

Catalase test

A biochemical test that determines if bacteria produce catalase, an enzyme that breaks down hydrogen peroxide into water and oxygen gas.

Oxidase test

A test used to determine if bacteria possess cytochrome c oxidase, indicated by the reagent turning purple.

Oxidative-fermentative (O-F) test

A test to determine if bacteria metabolize glucose by oxidation, fermentation, both, or neither by comparing sealed and unsealed tubes.

Phenol red broth

A medium used to determine if bacteria ferment a specific carbohydrate and produce gas as a byproduct.

Durham tube

A small inverted tube placed inside phenol red broth to trap gas produced during fermentation.

Citrate agar

A medium that turns from green to blue when bacteria produce alkaline byproducts while utilizing citrate.

Methyl red (MR) test

A test used to determine if bacteria perform mixed-acid fermentation, producing stable acidic end products.

Voges-Proskauer (VP) test

A test that detects acetoin, a neutral fermentation product, indicated by a red color change after adding reagents.

Urease test

A test that indicates if bacteria produce urease to hydrolyze urea into ammonia and carbon dioxide, turning the medium bright pink.

ASEPTIC TRANSFER

(Plate → Plate, Broth, or Slant)

Steps

Label the medium.

Sterilize the inoculating loop until red hot.

Allow the loop to cool.

Pick up the culture tube.

Remove the cap with your pinky finger.

Flame the tube opening.

Insert the cooled loop and obtain bacteria.

Flame the tube opening again.

Replace the cap.

Open the sterile medium only slightly.

Transfer the bacteria.

Close the medium immediately.

Flame the loop again.

Incubate as directed.

QUADRANT STREAK

Steps

Sterilize the loop.

Cool the loop.

Obtain a small amount of bacteria.

Streak Quadrant 1.

Flame and cool the loop.

Drag from Quadrant 1 into Quadrant 2.

Flame and cool again.

Drag into Quadrant 3.

Flame and cool again.

Drag into Quadrant 4.

Incubate the plate upside down.

SIMPLE STAIN

Steps

Label slide.

Add one drop of water (if using colonies from agar).

Transfer bacteria.

Prepare a thin smear.

Air dry completely.

Heat fix.

Flood with stain (methylene blue/crystal violet/safranin).

Wait approximately 1 minute.

Rinse gently with water.

Blot dry with bibulous paper.

Observe under oil immersion.

GRAM STAIN

Steps

Prepare smear.

Air dry.

Heat fix.

Apply crystal violet (1 minute).

Rinse.

Apply Gram's iodine (1 minute).

Rinse.

Apply alcohol/acetone decolorizer (10–20 seconds, or until runoff is nearly clear).

Immediately rinse.

Apply safranin (1 minute).

Rinse.

Blot dry.

Observe using the 100× oil immersion objective.

ENDOSPORE STAIN

Steps

Prepare smear.

Air dry.

Heat fix.

Place slide over steaming water.

Cover smear with paper.

Flood with malachite green.

Steam approximately 30 minutes, keeping the paper saturated with stain.

Remove paper.

Cool.

Rinse with water.

Counterstain with safranin (about 1 minute).

Rinse.

Blot dry.

Observe under oil immersion.

ACID-FAST STAIN

Steps

Prepare smear.

Heat fix.

Apply carbol fuchsin.

Steam gently.

Cool.

Rinse.

Decolorize with acid-alcohol.

Rinse.

Counterstain with methylene blue.

Rinse.

Blot dry.

Observe.

⭐ Positive

Red/pink cells.

⭐ Negative

Blue cells.

CAPSULE STAIN

Steps

Mix bacteria with Congo red.

Spread thinly.

Air dry only (do NOT heat fix).

Apply Maneval's stain.

Rinse gently if directed.

Observe.

⭐ Capsule

Clear halo.

CATALASE TEST

Steps

Place one drop of hydrogen peroxide on a clean slide.

Using a sterile wooden stick or loop (avoid blood agar colonies), transfer bacteria into the peroxide.

Observe immediately.

Positive

Immediate bubbles.

Negative

No bubbles.

OXIDASE TEST

Steps

Place oxidase reagent on filter paper (or use oxidase disk).

Transfer bacteria using a sterile wooden stick.

Observe within 10–20 seconds.

Positive

Purple.

Negative

No purple.

⭐ Don't wait too long—late color changes can be false positives.

PHENOL RED FERMENTATION

Steps

Inoculate phenol red broth containing a carbohydrate and Durham tube.

Incubate.

Observe color.

Observe gas.

Yellow

Acid.

Bubble

Gas.

Red

Negative.

Pink

Protein utilization.

O-F TEST

Steps

Inoculate two O-F tubes using a stab.

Cover one tube with sterile mineral oil.

Leave one tube open.

Incubate.

Compare colors.

Both yellow

Fermentative.

Only open yellow

Oxidative.

Neither yellow

Non-saccharolytic (does not use the carbohydrate).

CITRATE TEST

Steps

Lightly inoculate Simmons citrate slant.

Incubate.

Observe color and growth.

Positive

Blue with growth.

Negative

Green.

MR TEST

Steps

Inoculate MR-VP broth.

Incubate.

Add methyl red reagent.

Observe immediately.

Positive

Red.

Negative

Yellow/orange.

VP TEST

Steps

Use incubated MR-VP broth.

Add α-naphthol.

Add potassium hydroxide (KOH).

Mix gently.

Wait 15–30 minutes.

Positive

Red.

Negative

Copper/no red.

NITRATE TEST

Steps

Inoculate nitrate broth.

Incubate.

Add Reagent A.

Add Reagent B.

If red:

Positive.

If no red:

↓

Add zinc.

After zinc:

Red

↓

Negative.

Still no red

↓

Positive (nitrate reduced beyond nitrite).

UREASE TEST

Steps

Inoculate urea agar slant.

Incubate.

Observe color.

Positive

Bright pink.

Negative

Orange/yellow.

INDOLE TEST

Steps

Inoculate tryptone broth.

Incubate.

Add Kovac's reagent.

Observe the surface.

Positive

Red ring.

Negative

Yellow ring.

 PHENYLALANINE DEAMINASE (PAD)

Steps

Inoculate PAD slant.

Incubate.

Add ferric chloride.

Observe.

Positive

Green.

Negative

Yellow.

STARCH HYDROLYSIS

Steps

Streak starch agar.

Incubate.

Flood plate with iodine.

Observe.

Positive

Clear halo around growth.

Negative

Blue-black agar surrounding growth.

 BILE ESCULIN

Steps

Inoculate bile esculin slant.

Incubate.

Observe.

Positive

Blackening of the medium.

Negative

No blackening.

KIA (Kligler Iron Agar)

Steps

Stab the butt.

Streak the slant.

Incubate.

Observe:

Slant color

Butt color

Gas

H₂S (black precipitate)