Sample Processing

1. Why is sample processing necessary for naturally occurring microbial populations?

Because they are rarely found in concentrations convenient for direct measurement (either too many or too few).

2. Procedure used when there are too many microorganisms to count

Serial Dilution.

3. Goal of using a diluent in serial dilution

To bring the population to a quantifiable level without changing the original microbial counts.

4. Two purposes of specialized diluents

Resuscitation of stressed bacteria or recovery of specific groups.

5. Additive used when samples have high hydrocarbon or oil content to ensure mixing

Dispersant (e.g., Emulsifiers like Tween 80 or Polysorbate 20).

6. Tool used to distribute a sample throughout the diluent via high-speed vibration

Vortex mixer.

7. Two methods used when there are too few microorganisms to measure

Centrifugation or passage through membrane filters.

8. Application: You are testing 100 ml of water for Pseudomonas aeruginosa. Why must you use a filter?

Because regulatory requirements state it must be "negative in 100 ml," so you must concentrate the organisms from that large volume onto one filter for growth.

9. Importance of "Physiological Requirements" during processing

You must maintain specific conditions (e.g., high heat for thermophiles) during lab work to keep the target organisms alive.

10. Efficiency of microbial recovery depends on these five factors

Choice of diluent, time of mixing, temperature, dispersant used, and degree of agitation.

11. Term for organisms that are alive but cannot be grown on standard laboratory media

Viable But Non-Culturable (VBNC).

12. Why are some organisms "non-culturable"?

They may have hard-to-replicate growth requirements or obligate symbiotic relationships with hosts. (Some are dormant, others are non viable, others are VBNC)

13.

14. Approach where organisms must be recovered and grown from environmental samples

Culture-Dependent Approach.

15. Purpose of dilution-plating in a culture-dependent approach

To separate aggregates of microorganisms so individual units can reproduce and form visible, countable growth.

16. Method used to selectively grow a specific group of microorganisms by using inhibitory substances or limiting conditions

Enrichment culture.

17. A classic model system used for growing anaerobic photosynthetic and sulfate-reducing bacteria

Winogradsky Column.

18. Observable traits of an organism produced by the interaction of its genotype and the environment

Phenotype.

19. Main disadvantage of phenotypic detection

It is useless for non-culturable organisms because they must express traits during in vitro culture. In addition to this, It must be culturable, have established growth parameters, and have known biochemical profiles.

20. Three categories of characteristics observed in phenotypic detection

Morphological, Colonial/Cultural, and Physiological/Metabolic.

21. Why is the Winogradsky column considered an "enrichment" method?

Because it creates various micro-environments (oxygen and sulfide gradients) that naturally select for specific groups like purple and green sulfur bacteria.

22. If you use a vortex mixer too aggressively on a sample containing delicate protozoa, what might happen to your count?

The count will decrease because the mechanical agitation may rupture or kill the delicate cells.

23.

24. Staining technique used specifically for the Domain Bacteria to differentiate cell wall types

Gram Staining.

25. Specific staining method used to visualize bacterial appendages used for motility

Bailey’s Flagellar Staining.

26. Type of staining used to identify genera like Bacillus and Clostridium that produce dormant, resistant structures

Endospore Staining.

27. Observation of microorganisms grown on standard laboratory media under suitable conditions

Cultural/Colonial Morphology.

28.

29. Genetic elements used to track specific microbial populations or biological functions in the environment

Reporter Genes (Bioreporters).

30. Definition of a "Genetic Marker"

A genetic element that permits the detection of an unrelated biological function.

31. List four common examples of reporter genes

lacZ gene, xylE gene, lux gene, and Green Fluorescent Protein (GFP).

32. Specific reporter gene used in Pseudomonas that encodes catechol 2,3-dioxygenase

C23O, It produces a yellow color (2-hydroxymuconic semialdehyde) from the degradation of catechol.

33.

34. Classification based on the distribution of specific chemical components in the cell

Chemotaxonomy.

35. List five types of chemical markers used in microbial classification

Lipids, cell wall amino acids/sugars, sterols/hopanoids, quinones, and cytochromes.

36. Examples of organisms known for having "High GC" DNA base composition

Actinomycetes and Streptomycetes.

37. Example of a genus known for "Low GC" DNA base composition

Clostridium.

38. Analysis that uses the unique pattern of lipid composition to identify microbial species

Lipid Profile Analysis.

39. Identification method that utilizes liquid AP-MALDI to show unique spectra for different species

Lipid Profiling (Mass Spectrometry).

40.

41. What does the acronym FAME stand for?

Fatty Acid Methyl Ester Analysis.

42. Primary analytical tool used to identify and quantify fatty acids in FAME analysis

Gas Chromatography (GC).

43. Why is "Methylation" performed during FAME analysis?

To make the fatty acids more volatile, which is required for gas chromatography.

44. The specific signature fatty acid "10 Me 16:0" is a marker for which genus?

Desulfovibrio.

45. What are the five structural features used to identify "Signature Fatty Acids"?

Chain length, double bonds, rings, branches, and hydroxyl (OH) groups

46. Component detected to estimate total bacterial biomass

Palmitic acid (16:0).

47. Signature fatty acids for microeukaryotes

Polyunsaturated fatty acids.

48. Signature fatty acids for aerobic prokaryotes

Monounsaturated acids.

49. Signature fatty acids for Gram-positive anaerobes

Saturated and branched fatty acids (C14–C16).

50. Signature fatty acids for Sulfate-Reducing Bacteria (SRB)

Saturated and branched fatty acids (C16–C19).

51. Questions that should be answered in sample processing

who are there, how many are there (number, mass, activity)

52. are studying a Pseudomonas strain in soil and need to know if it is actively degrading a pollutant. Which reporter gene system would allow you to see a yellow color change?

The C23O (catechol 2,3-dioxygenase) system.

53. Critical Thinking: Why is FAME analysis considered more "comprehensive" than Gram staining?

Gram staining only divides bacteria into two groups, while FAME provides a specific "chemical fingerprint" that can identify organisms down to the species or strain level.

54. Application: You detect a high concentration of polyunsaturated fatty acids in a water sample. What group of organisms is likely dominant?

Microeukaryotes.

55. True or False: Hopanoids and sterols are used as chemotaxonomic markers because they are found in every single living cell.

FALSE; they are used because their specific distribution varies among different groups, aiding in classification.