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. Make 2nd streak (after sterilizing)
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 g
Peptone 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, 100ºC)
Agar
Does not resolidify until 42ºC

28. Formula
Difco™ Nutrient Agar
Approximate Formula* Per Liter
Beef Extract g
Peptone g
Agar 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

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

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

51. Gram Stain Hans Christian Gram Most widely used staining
Based on the ability of a bacterium to absorb and retain a stain due to cell wall structure
Gram Positive cells have a thick layer of peptidoglycan. Stain is better absorbed and retained in this
Gram negative cells have a thin layer of peptidoglycan so they are easily decolorized by alcohol. Need to be counterstained with safranin to view.

53. Overview of Gram-staining process:
Crystal Violet
Iodine
Ethanol
Safranin
Gram+
Gram–
Results:

54. Escherichia coli
Staphylococcus epidermis
Bacillus cereus

55. Gram Positive
Gram Negative

56. Endospore staining
Endospores are highly resistant, thick walled structures.
They are highly resistant to:
radiation
chemical agents
extremely high temperatures
drying out
and other normally harmful environments. 

57. Malachite green needs to be heat driven into fixed stain.
Bacillus and Clostridium are the two most common endospore-producing genera. 
Due to the highly resistant nature of endospores, it is necessary to steam stain into them. 
Most common endospore staining technique is the Schaeffer-Fulton method.
Malachite green needs to be heat driven into fixed stain.
Counter stain with Safranin

58. Endospore Formation

59. Overview of endospore-staining process:
Heat + Malachite Green
Water
Safranin
Vegatative cell
Cell with Endospore
Results:

60. Schaeffer-Fulton method
Using aseptic technique, prepare a bacterial smear on a clean slide, air dry and gently heat fix.
Prepare a boiling water bath.
Cover the slide with a piece of paper towel, and place on a staining rack over the water bath.
Flood the paper towel on the slide with Malachite green (primary stain).
Steam the slide for ten minutes.
Remove the slide from the water bath, and remove the paper towel
Allow the slide to cool, and then rinse with deionized water until the water runs clear
Pour off any excess water and apply Safranin (counterstain) for two minutes.
Rinse excess Safranin off with deionized water, and blot the slide dry with bibulous paper.
Examine the slide with a light microscope under oil immersion.

61. How it works

62. Staphylococcus epidermis
Bacillus cereus

63. B. subtilis
B. cereus

64. Position of Endospores
Central
(1, 4) Central endospore
(2, 3, 5) terminal endospore,
(6) lateral endospore
Terminal
Subterminal

65. Acid Fast Stain
Used with strains that are difficult to stain due to glycolipids in the cell wall.
Mycobacterium (TB & pneumonia)
Use basic fuchsin (carbol fuchsin) with heat to fix slide then acid - alcohol to decolorize.
Second staining w/ methylene blue
for non-acid fast bacteria.

66. Overview of acid-fast staining process:
Results:
Heat + Carbol Fuchsin
Acid alcohol
Methylene blue
Acid-Fast
Non-AF
Acid-fast rods

67. Blue is Micrococcus luteus and pink is Mycobacterium smegmatis

68. Mix of acid-fast & non-acid-fast bacteria (Mycobacterium gordonae & Staphylococcus epidermidis)