Tools of the Laboratory

1. Tools of the Laboratory Chapter 3

2. THE 5 I’S • Of culturing • Goal: grow, examine, and characterize microorganisms • Inoculation • Incubation • Isolation • Inspection • Identification

3. 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?

4. Incubation • Goal: Produce growing environment • Important factors • Temperature (20-40ºC) • O2/CO2 content • Pressure • Can take hoursweeks to produce culture

5. 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

6. Pure culture • AKA “axenic” culture • Only one species or type on the plate • Usually what we want • Analogy: only carrots in a garden

7. 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

8. Mixed culture • 2+ easily differentiated species • Analogy: peas and carrots in a garden

9. Contaminated Culture • Unwanted microbes have somehow gotten in • NEVER want this!! • Analogy: Weeds in your garden

12. Isolation • Goal: get one type/strain/species of bacteria by itself • Must dilute bacteria first • Want single, isolated colonies

13. Streak Plate Method • Most common method • With sterile inoculating loop, spread sample over entire media surface • Thins sample over several sections

14. Sterilize Loop Pick up desired colony Make 1st streak Make 2nd streak (after sterilizing) Make 3rd streak (after sterilizing) Make 4th streak (after sterilizing)

16. Pour Plate Method • Sample is added to plate and then liquid agar is added • Swirled to mix • Colonies grow on top and in medium

17. Spread plate technique • Small liquid volume of sample is transferred to plate • Spread evenly with “hockey stick”

18. Inspection • Macroscopically and microscopically • Staining may be used here • May have to isolate again

19. Identification • Ok, so what is it? • Can use appearance, but not as easy as you’d think

20. Identification • Nutrition requirements • Metabolism products • Enzymes • Energy mechanisms • Antibiotic resistance • DNA • Host response

21. Bacteria Isolation Practical Lab

22. 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

23. Media • What we grow bacteria in/on • Characteristics • Physical state • Chemical composition • Functional types

24. 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.

25. Semi-solid media • At room temp, clot-like consistency • Contains some agar/gelatin • Used in motility tests

26. 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

27. Solid Media • Liquefiable solid media • Aka reversible • Changes state with temp (solid at room temp, melts @ 100ºC) • Agar • Does not resolidify until 42ºC

28. 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.

29. Chemical content • Either synthetic or nonsynthetic • Synthetic • Chemically defined • Exact formula • Research and cell culturing • Can be 50+ ingredients!

30. Chemical COntent • Either synthetic or nonsynthetic • Nonsynthetic • Aka “complex” • No exact formula • Extracts/parts from plants/animals/yeast • Uses blood, serum, meat, milk

31. Media Functions • Some microbes can NEVER be cultured • But for those that can… • General purpose media • Enriched media • Selective media • Differential media

32. General purpose media • Grows many typical microbes • Often a nonsynthetic media • Ex: nutrient agar and broth

33. Enriched media • Contains extra nutrients that certain species need to survive • “Picky” bacteria are called fastidious • EX: blood agar for fastidious streptococci

34. 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

36. 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

39. Intro to Selective media Lab

40. Smear, Heat Fix, and simple stain Procedure https://www.youtube.com/watch?v=8ODeT9DLHKI

41. 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

42. Staining • Bacteria are not naturally vividly colored • Difficult to see

43. Simple stains • Contain only 1 dye • Crystal violet, Methylene blue, Malachite green, safranin • Apply to a fixed slide & then flood with stain and rinse.

44. Positive stain • More common • Basic dyes – methylene blue, crystal violet dye DNA/RNA • Acid dyes – acid fushin, congo red stain proteins

45. 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

47. Negative stain video • https://www.youtube.com/watch?v=avveXgPWVJ8

48. 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)

49. Hematoxylin and eosin stain

50. 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