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Tools of the Laboratory. Chapter 3. THE 5 I ’ S. Of culturing Goal: grow, examine, and characterize microorganisms Inoculation Incubation Isolation Inspection Identification. Inoculation. Goal: Produce a culture Introduce a tiny sample to a nutrient medium
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Tools of the Laboratory Chapter 3
THE 5 I’S • Of culturing • Goal: grow, examine, and characterize microorganisms • Inoculation • Incubation • Isolation • Inspection • Identification
Inoculation • Goal: Produce a culture • Introduce a tiny sample to a nutrient medium • Only want 1 microorganism type/sample • Where could you get a sample to inoculant?
Incubation • Goal: Produce growing environment • Important factors • Temperature (20-40ºC) • O2/CO2 content • Pressure • Can take hoursweeks to produce culture
Cultures • A culture is when microbes are growing enough on a culture so that you can see them • Culture vocab • Pure culture • Subculture • Mixed culture • Contaminated culture
Pure culture • AKA “axenic” culture • Only one species or type on the plate • Usually what we want • Analogy: only carrots in a garden
Subculture • Can lead to a pure culture • Inoculate a plate from an old plate with a well-isolated colony • Analogy: using seeds from carrot to start new garden
Mixed culture • 2+ easily differentiated species • Analogy: peas and carrots in a garden
Contaminated Culture • Unwanted microbes have somehow gotten in • NEVER want this!! • Analogy: Weeds in your garden
Isolation • Goal: get one type/strain/species of bacteria by itself • Must dilute bacteria first • Want single, isolated colonies
Streak Plate Method • Most common method • With sterile inoculating loop, spread sample over entire media surface • Thins sample over several sections
Sterilize Loop Pick up desired colony Make 1st streak Make 2nd streak (after sterilizing) Make 3rd streak (after sterilizing) Make 4th streak (after sterilizing)
Pour Plate Method • Sample is added to plate and then liquid agar is added • Swirled to mix • Colonies grow on top and in medium
Spread plate technique • Small liquid volume of sample is transferred to plate • Spread evenly with “hockey stick”
Inspection • Macroscopically and microscopically • Staining may be used here • May have to isolate again
Identification • Ok, so what is it? • Can use appearance, but not as easy as you’d think
Identification • Nutrition requirements • Metabolism products • Enzymes • Energy mechanisms • Antibiotic resistance • DNA • Host response
Review • What are the 5 I’s of culturing? • What are the differences between • Pure culture • Mixed culture • Contaminated culture • Subcultures • What are the differences between the following: • Streak plate method • Pour Plate method • Spread Plate method
Media • What we grow bacteria in/on • Characteristics • Physical state • Chemical composition • Functional types
Physical state • Liquid media- water based sol’n that do not solidify above freezing • Aka broths, milks, infusions • Ex: nutrient broth beef extract and peptone in H2O Difco™ Nutrient Broth Approximate Formula* Per Liter Beef Extract ................................................................ 3.0 g Peptone ..................................................................... 5.0 g *Adjusted and/or supplemented as required to meet performance criteria. Directions for Preparation from Dehydrated Product 1. Dissolve 8 g of the powder in 1 L of purified water. 2. Autoclave at 121°C for 15 minutes. 3. Test samples of the finished product for performance using stable, typical control cultures.
Semi-solid media • At room temp, clot-like consistency • Contains some agar/gelatin • Used in motility tests
Solid Media • Firm substance for cells to grow on • Can be liquefiable or nonliquefiable • Nonliquefiable—cannot be melted • Less versatile • Remains solid after heating • Could be potato slice or proteins that denature
Solid Media • Liquefiable solid media • Aka reversible • Changes state with temp (solid at room temp, melts @ 100ºC) • Agar • Does not resolidify until 42ºC
Formula Difco™ Nutrient Agar Approximate Formula* Per Liter Beef Extract ................................................................ 3.0 g Peptone ..................................................................... 5.0 g Agar ......................................................................... 15.0 g *Adjusted and/or supplemented as required to meet performance criteria. Directions for Preparation from Dehydrated Product 1. Suspend 23 g of the powder in 1 L of purified water. Mix thoroughly. 2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. 3. Autoclave at 121°C for 15 minutes. 4. Test samples of the finished product for performance using stable, typical control cultures.
Chemical content • Either synthetic or nonsynthetic • Synthetic • Chemically defined • Exact formula • Research and cell culturing • Can be 50+ ingredients!
Chemical COntent • Either synthetic or nonsynthetic • Nonsynthetic • Aka “complex” • No exact formula • Extracts/parts from plants/animals/yeast • Uses blood, serum, meat, milk
Media Functions • Some microbes can NEVER be cultured • But for those that can… • General purpose media • Enriched media • Selective media • Differential media
General purpose media • Grows many typical microbes • Often a nonsynthetic media • Ex: nutrient agar and broth
Enriched media • Contains extra nutrients that certain species need to survive • “Picky” bacteria are called fastidious • EX: blood agar for fastidious streptococci
Selective media • Contains 1+ agents that inhibit growth of certain microbe(s) • “selects” for one type of bacteria • Helps isolate it • Ex: Enterococcus faecalis broth
Differential media • Grow several types of microbes but brings out visible differences • Colony size/color • Media color • Formation of gas bubbles/precipitate • pH dyes very effective
Smear, Heat Fix, and simple stain Procedure https://www.youtube.com/watch?v=8ODeT9DLHKI
Smears and stains • Robert Koch • A smear places microbes on a slide • Thin film, let air dry • Often fixed with heat • Kills and secures specimen
Staining • Bacteria are not naturally vividly colored • Difficult to see
Simple stains • Contain only 1 dye • Crystal violet, Methylene blue, Malachite green, safranin • Apply to a fixed slide & then flood with stain and rinse.
Positive stain • More common • Basic dyes – methylene blue, crystal violet dye DNA/RNA • Acid dyes – acid fushin, congo red stain proteins
Negative stains • Stain cannot penetrate the cell wall. Is repelled. • Background takes on stain. • No heat fixing or rinsing. Just a smear • Used on living cells to observe surface structures • Eosin, Nigrosin, India Ink
Negative stain video • https://www.youtube.com/watch?v=avveXgPWVJ8
Differential Stain • 2 different colored dyes • Primary dye and a counterstain • Difference in surface structure/chemistry determines staining properties • Red v. purple (Gram stain) • Red v. green (endospore) • Pink v. blue (acid fast)
Structural Stains • Emphasize special cell parts • EX: capsule stain (often done with India ink) • Ex: flagellar stain-deposits coat on skinny flagella and then stains it