BACT LAB: CULTURE & CULTURE MEDIA

CULTURE

Growth of microorganism (lots of colonies) in a culture media

CULTURE MEDIA

Mixture of nutrients such as:

• Carbon

• Nitrogen

• Phosphorus

• Hydrogen

• Oxygen

• Buffers

PURE CULTURE

Composed of only 1 species and the goal in bacteriology

MIXED CULTURE

Composed of more than 1 species

STOCK CULTURE

Several culture species contained in separate culture medium (1 species per culture medium)

• used for academic & industrial purposes

LIQUID MEDIUM

0% agar; allows growth of aerobes, Also known as “broth”

• Brain heart infusion

• Trypticase soy broth (TSB)

• Thioglycollate

SEMI-SOLID MEDIUM

0.5 - 1% agar; observe bacterial motility and detect presence of indole and sulfide production

• Sulfide indole motility medium

COLONY

used to describe a single isolated microorganism in a culture medium

TURBIDITY

Indication of bacterial growth in liquid medium becuase you can’t see colonies

18 - 24 HOURS

This is the average time of using liquid medium as an enrichment broth (to propagate microorganisms)

In semi-solid medium, a microorganism is considered motile when there is __________ outside of the puncture area.

DISPERSION / SIGN OF GROWTH

SOLID MEDIUM

2 - 3% agar; is always plated

• Triple sugar iron agar (TSI)

• MacConkey agar (MAC)

• Blood agar plate (BAP)

• Chocolate agar plate (CAP)

SYNTHETIC / DEFINED MEDIUM

• Known components

• Research purposes

• Preferred for isolation of cyanobacterium & chemoorganotrophs

  • BG-11 medium

NON-SYNTHETIC / COMPLEX MEDIUM

• Unknown substances

  • Peptones

  • Meat

  • Yeast extract

• Isolation of medically significant bacteria

  • Nutrient broth medium (NB)

  • TSB

  • MAC

TISSUE CULTURE MEDIUM

Used for obligate intracellular bacteria

• Rickettsia

• Chlamydia

HeLa 299 cells

Human cervical tissue

McCoy cells

Fibroblast

W138 cells.

Fibroblast.

PLATE MEDIA

Distribued into petri dish or plate

TUBE MEDIA

Prepared as butt, slant, or both butt and slant

• TSI

• SIM

• Simmon’s citrate agar

• Lysine iron agar (LIA)

STAB

Distribution method for butt, uses needle

• ANAEROBIC

FISH TAIL METHOD

Distribution method for slant, uses inoculating loop

• AEROBIC

NUTRITIVE MEDIA

• Routinely used w/o supplements

• Supports growth of most non-fastidious bacteria

• Meat & Soy bean extract

  • Nutrient agar (NA)

  • NB

  • TSB

ENRICHMENT MEDIA

• Propagate certain group of bacteria from mixed culture

• Contains specific nutrients w/o additional supplements

  • Alkaline peptone water (vibrio)

  • Selenite F. (salmonella)

  • Thioglycollate

  • Tetrathionate

  • Gram-neg broth

  • Lim broth (Streptococcus agalactaea)

The RBC in a blood agar plate should be ________ to indicate intact cellular structure.

INTACT

The RBC in a chocolate agar plate should be __________ for hemoglobin and NAD.

LYSED

SHEEP’s BLOOD

Your first choice of blood when preparing a BAP

BAP BLOOD CHOICES

1. Sheep

2. Horse

3. Human

Your first choice of blood when preparing a BAP from a human should be __________.

O+

DIFFERENTIAL MEDIA

Allow visualization of metabolic differences between groups of bacteria

• MAC

• BAP

• Eosin methylene blue (EMB)

• Hektoen enteric agar (HEA)

• Mannitol salt agar (MSA)

PINK

Color of organism when it has the ability to ferment lactose (MAC)

COLORLESS

Color of organism when it doesn’t have the ability to ferment lactose (MAC)

ALPHA HEMOLYSIS

Partial hemolysis w/ greenish coloration

• Escherichia coli

BETA HEMOLYSIS

Complete hemolysis w/ clearing of blood

• Streptococcus pyogenes

GAMMA HEMOLYSIS

No hemolysis

• Staphylococcus epidermidis

SELECTIVE MEDIA

Incorporated w/ antibiotics, dyes, or chemicals to inhibit growth of other organisms

• HEA

• MAC

• Xylose lysine deoxycholate agar (XLD)

• Bismuth sulfate agar (BSA)

• MSA

• Thayer-Martin agar (TMA)

GENTAMICIN BLOOD AGAR

Streptococcus

BACITRACIN CHOCOLATE AGAR

Haemophilus

BAP W/ AMPICILLIN

Aeromonas

PHENYLETHYL ALCOHOL

Gram + bacteria

COLISTIN-NALIDIXIC ACID AGAR (CNA).

Gram + bacteria.

INHIBITORY SUBSTANCE FOR GRAM ( + ) BACTERIA

• Crystal/Gentian violet

• Basic/Carbol fuchsin

• Bile salt

INHIBITORY SUBSTANCE FOR GRAM ( - ) BACTERIA

• Potassium tellurite

• Sodium azide

INHIBITORY SUBSTANCE FOR SWARMING BACTERIA

• Alcohol

• Chloral hydrate

A SWARMING BACTERIA SPECIES

Proteus spp.

HEKTOEN ENTERIC AGAR (HEA)

• Inhibits indigenous microbiota of the lower gastrointestinal tract (LGIT)

• Recovery of fecal bacteria

• Contains Bile salt & dyes

• pH indicator = Bromthymol blue

MACCONKEY AGAR (MAC)

• Inhibits gram ( + ) bacteria

• Recovery of fecal bacteria

• Contains Bile salts & Crystal violet

XYLOSE LYSINE DEOXYCHOLATE AGAR (XLD)

• For fecal bacteria

• Differentiates Shigella & Salmonella

• Contains:

  • Xylose

  • Lysine

  • Sucrose

  • 0.25% sodium deoxycholate & sodium thiosulfate

MANNITOL SALT AGAR (MSA)

Supports growth of Staphylococcus aureus (a halophile)

THAYER-MARTIN AGAR (TMA)

Selective for Neisseria sp.

SPECIAL MEDIA

Isolate bacteria w/ specific growth requirements

• Lowenstein-Jensen medium (LJ)

• Thiosulfate citrate-bile salts-sucrose agar (TCBS)

BLOOD AGAR PLATE (BAP)

Differentiate hemolytic pattern of bacteria

CHOCOLATE AGAR PLATE (CAP)

Recovery of Haemophilus

STEPS IN MEDIA PREP (PLATES)

1. Weigh

2. Dissolve (distilled H2O)

3. Sterilize (autoclave)

4. Dispense

STEPS IN MEDIA PREP (TUBES)

1. Weigh

2. Dissolve

3. Dispense

4. Sterilize

Set the autoclave to ______ psi for ______ minutes at ______° C.

15 psi; 15 minutes; 121° C.

Aseptically dispense approx. ____ ___ of agar in sterile petri plates

25 mL

Solve for the agar amount needed for 20 plates

Given: suspend 40g in 1,500mL

13.33g