Laboratory Procedures

1. Laboratory Procedures

2. Bacteria

3. Culturing Bacteria • Culturing: To introduce a specific microorganism into a solid or liquid broth in order to grow that microorganism for laboratory testing. • Four steps: • Swabbing the surface/patient you want to culture. • Streaking the swab onto an agar plate. • Staining/testing for identification. • Maintaining a stock culture.

4. Culturing A Patient • Proper Method: • Place cotton swab against area being cultured. • Gently spin the swab or move side-to-side to maximize number of microorganisms picked up. • Lift swab away from surface and then remove from area.

5. Streaking Onto Agar • The first step we must take is to learn about agar.

6. Agar • Agar: A complex polysaccharide medium used to grow microorganisms. • First used by Koch when developing his postulates, but still used to this day! • Begins as a solid, but can be melted into a liquid. • Once melted, it does not solidify until it cools to 40˚C/104˚F. • Allows heat-sensitive substances to be added just before solidification.

7. Agar • Basic agar is made by combining either chicken or beef broth with a base agar powder. • Some types of agar go father than this! • Peptone: A product of enzyme digestion of proteins easily utilized by microorganisms – a common ingredient!

8. Agar • Bacteria divides exponentially on either solid or liquid agar. • Liquid agar provides a mixed culture populated by several bacteria. • Solid agar provides growth in colonies that are decedents of a single cell.

9. Streaking Onto Agar • The second step is to figure out which type of agar you want to use, based on patient symptoms.

10. Classes of Agar • Synthetic Media: A medium prepared in the laboratory from materials with a precise or reasonably well-defined composition. • Complex Media: A medium with a similar base composition as synthetic media, but with slightly differing composition from batch to batch. • E.g.: Could contain beef, yeast or soy protein, in any combination.

11. Classes of Agar • Selective Medium: Encourages the growth of some bacteria but discourages the growth of others. • Typically involves including certain antibiotics that will not harm the particular bacterium it is designed to grow. • Used to try to grow just one type of organism.

12. Classes of Agar • Differential Medium: Contains a constituent that causes an observable change. • Typically has chemicals that will induce a chemical reaction and color change when certain organisms are present. • This lets us tell different colonies apart based on color change. • Used to tell things apart!

13. Classes of Agar • Enrichment Medium: Contains special ingredients to allow a particular organism to flourish. • Especially useful when there may not be enough organisms to be isolated and identified using a normal agar method. • Problem: It does not suppress the growth of other organisms – EVERYTHING goes wild!

14. Types of Agar – Blood Agar • Defining Feature: • Contains 5% blood cells from an animal, usually sheep. • What Will Grow: • Most bacteria will grow on this.

15. Types of Agar – Chocolate Agar • Defining Features: • Made of lysed red blood cells, typically sheep. • Nutrient medium used to culture fastidious organisms. • DOES NOT give aemolysis data

16. Types of Agar – Chocolate Agar • What Will Grow: Most bacteria will grow on this. • Haemophilus grows due to X & V factors from the sheep blood – cannot differentiate between the species though! • Neisseria grows due to nutrient-rich environment.

17. Types of Agar – Luria Bertani (LB) Agar • Defining Feature: • A subtype of nutrient agar. • General medium used for routine cultivation. • What Will Grow: • Does not grow fastidious microorganisms. • Does not preferentially grow one kind of bacteria over another. • Grows everything!

18. Types of Agar – Miller’s LB Agar • Defining Feature: • A variation of LB agar. • Contains the same basic components of LB agar, but in different proportions. • What Will Grow: • Only Gram Negative bacteria will grow.

19. Types of Agar – MacConkey Agar • Defining Feature: • E. coli differentiates by growing into red colonies due to sugar fermentation. • Can be mixed with or without sugar lactose. • What Will Grow: • Will only grow Gram Negative bacteria.

20. Types of Agar – Neomycin Agar • Defining Feature: • Contains neomycin (antibiotic). • Used to culture anaerobic microorganisms. • What Will Grow: • Only species not sensitive to neomycin will grow. • Stops the growth of Gram Negative bacilli and staphylococci. • Allows streptococcus species to grow abundantly.

21. Types of Agar – Neomycin Agar • Interesting Fact: Neomycin was discovered in 1949 by the microbiologist Selman Waksman. P • Produced naturally by the bacterium Streptomyces fradiae. • Has a broad spectrum of effects, killing both gram-positive and gram negative bacteria. • Relatively toxic to humans, and some people have allergic reactions to it.

22. Types of Agar – Non-Nutrient Agar • Defining Feature: • May be used to grow non-bacterium microorganisms. • What Will Grow: • Does not grow bacteria. • May be used for growing other microorganisms.

23. Types of Agar – Trypticase Soy Agar (TSA) • Defining Feature: • Grows the widest variety of microbes. • Uses beef broth. • Contains some yeast extracts. • What Will Grow: • Will grow bacteria & fungi. • Doesn’t grow all bacterium – some find it too rich, others find it deficient.

24. Types of Agar – Sabouraud Agar • Defining Feature: • Used to grow fungi. • Has a low pH (kills most bacteria). • Contains gentamicin antibiotic (particularly useful for Gram Negative bacteria). • What Will Grow: • Grows fungi.

25. Types of Agar – Thayer-Martin Agar • Defining Feature: • Designed to isolate Neisseria gonorrhoeae. • What Will Grow: • Specifically designed for Neisseria gonorrhoeae.

26. Types of Agar – Tryptic Soy Agar (TSA) • Defining Feature: • A basic medium for general culturing. • Mainly used as an initial growth medium for… • Observing colony morphology. • Developing pure cultures. • Gaining sufficient growth for biochemical testing. • Culture storage. • What Will Grow: • Just about everything!

27. Types of Agar – Xylose Lysine Deoxychocolate Agar • Defining Feature: • Used for culturing stool samples. • Contains two different indicators. • Colonies of organism that ferment lactose will appear yellow. • What Will Grow: • Formulated to inhibit Gram-positive bacteria. • Formulated to encourage Gram-negative bacilli.

28. Streaking • Streaking: The specific technique for isolating bacterium on solid agar. • Streak Plate Method: A method used to obtain colonies from a single parent species.

29. Streak Plate Method 1. The streaking tool is held in open flame to sterilize, then dipped into a colony or culture. • In the case of a patient culture, it is applied directly to the agar. 2. The culture is moved in a back-and-forth motion across the top of the plate.

30. Streak Plate Method 3. The plate is turned 90˚ and a sterile loop is used to streak down from one corner, only touching the previously streaked region once, then continuing up and down the far side of the plate. 4. The plate is turned 90˚ again, and a sterile loop is used to repeat the streaking process.

31. Staining For Identification • Staining serves several purposes… • Tells us information about the cell wall. • Helps us to identify the microorganism. • Helps us to figure out what chemotherapeutic agents will kill it.

32. Types of Stains • Stains and dyes are molecules that bind to a cellular structure to give it color. • Cationic Dyes: Basic, positively charged dyes that are attracted to negatively charged cell components. • Includes methylene blue, crystal violet, safranin, & melachite green. • Anionic Dyes: Negatively charged dyes that are attracted to positively charged material. • Includes eosin & pictric acid.

33. Methods of Staining • Simple Stain: Uses a single dye. • Allows us to determine cell shape and arrangement under a microscope. • Can be done with methylene blue, safranin, crystal violet, and carbolfuchsin. • Differential Stain: Uses two or more dyes. • Allows us to distinguish between the different parts of an organism and between organisms close to each other. • Examples: Gram Stains and Ziel-Neelsen Acid-Fast Stain.

34. Gram Stain • Gram Stain: A staining method that allows us to determine the type of cell wall a bacterium possesses. • The bacteria are streaked onto a glass slide. • The slide is passed over a flame to heat-fix the material. • The slide is flooded with crystal violet (purple) for 15 seconds. • The slide is rinsed with iodine, then water. • The slide is flooded with safranin (red) for 15 seconds. • The slide is light rinsed with water and pat dry.

35. Gram Stain

36. Gram Stain • Reminder: • Gram-Positive & Acid-Fast bacteria retain the crystal violet due to the thickness of their cell walls. • Gram-Negative bacteria loose the crystal violet and keep the safrinin (since no alcohol rinse is used to remove it!) due to the thinness of their cell wall. • Gram-Variable cells stain purple and red – usually due to the cells being too old when cultured, which decreases their ability to retain stains. • This is a differential stain.

37. Gram Stain

38. Ziehl-Neelsen Acid-Fast Stain • Ziehl-Neelsen Acid-Fast Stain: Tests for the high lipid content of their cell walls. • The same basic procedures are used as in Gram Staining, except… • Crystal violet is replaced with carbolfuchsin (a red stain). • Safranin is replaced with a blue dye. • Acid-Fast bacteria stains red from the carbolfuchsin. • Bacteria that stains blue should be Gram stained. • This is a differential stain.

39. Ziehl-Neelsen Acid-Fast Stain • Tuberculosis:

40. Flagellar Stain • Used to identify specific structures within an organism. • Specifically, flagellated bacteria appear dark with flagella appearing as silver or red lines. • This is a special stain.

41. Stock Culture • Pure Culture: A culture containing only one species of organism. • Streak Plate Method is used to obtain single-species colonies. • Stock Culture: A culture grown in liquid broth from one organism or one colony of organisms. • This can be innoculated onto fresh agar when the bacteria is needed for study. • Aseptic Techniques and other measures must be used to prevent contamination of the stock culture.

42. Measuring Bacterial Growth • Turbidity: The cloudy appearance of a liquid broth with containing bacterial colonies.

43. Measuring Bacterial Growth • Spechtophotometer: An instrument used to measure the degree of light transmission through a turbid culture. • Allows an indirect measurement of the number of cells present in the culture. • Allows measurement without disturbing the culture. • Can be inaccurate if there are too many or not enough overall cells.

44. Measuring Bacterial Growth • Standard Plate Count: Estimates the number of bacteria from a culture assuming that each colony grown on a solid agar plate originated from a single organism.

45. Measuring Bacterial Growth • Serial Dilutions: A method of diluting a liquid culture to prevent over-crowding of colonies when grown on solid agar. • Multiplying the number of colonies by the ratio of dilution to estimate the number of bacterial cells in the original culture. • For example: If a microbiologists dilutes a broth to 1:1000 ratio of broth to dilutant, and 50 colonies grow on agar by the next day, he can estimate the 50,000 bacterial cells per one milliliter of original culture.

46. Measuring Bacterial Growth • Most Probable Number (MPN): An estimation of the number of bacteria that is used when there are too few cells to use other methods.

47. Measuring Bacterial Growth • Most Probable Number (MPN): • Measures the bacterial waste byproducts produced. • A small glass tube is inverted & placed into the test tube containing liquid culture so that it fills with broth. • Gas produced by the fermentation of bacteria will be trapped in the inverted tube. • The gas can then be compared to a standardized curve to estimate the number of organisms present in the sample.

48. Filtration • Filtration: Forces a known volume of liquid or air through a filter with pores too small for bacteria to pass through. • The filter can then be placed on solid media and allowed to grow. • The resulting colonies can then be counted using standard plate count. • The standard plate count allows an estimate of the number of organisms per liter of air or water to be estimated.

49. Fungi

50. Culturing Fungi • Fungi can be grown, cultured and identified using similar laboratory methods to those used on bacteria! • Acidic, high-sugar media with antibiotics to prevent bacterial growth can be used. • Most pathogenic fungi take two to four weeks to grow as much as bacteria do in a 24-hour period. • This leads to slow identification!